Polypeptides with xylanase activity

ABSTRACT

Polypeptides with xylanase activity modified to increase bran solubilization and/or xylanase activity. The modification comprises at least an amino acid modification i position 110 and may have further modifications of one or more amino acids in position 11, 12, 13, 34, 54, 77, 81, 99, 104, 113, 114, 118, 122, 141, 154, 159, 162, 164, 166 or 175 wherein the positions are determined as the position corresponding the position of  Bacillus subtilis  xylanase (SEQ ID NO 1) The polypeptides have at least 88% identity with SEQ ID NO 1 or 75% identity to a sequence selected from SEQ ID NO 2-22.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationPCT/DKP2009/050351 filed Dec. 23, 2009, which designates the U.S. andwas published by the International Bureau in English on Jul. 1, 2010,and which claims the benefit of European Patent Application No.08172749.7, filed Dec. 23, 2008, and U.S. Provisional Application No.61/146,155, filed Jan. 21, 2009, all of which are hereby incorporatedherein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to polypeptides with xylanase activity anduses thereof. The present invention also relates to method of modifyingpolypeptides with xylanase activity to affect, preferably to increase,xylanase activity and/or bran solubility.

BACKGROUND OF THE INVENTION

For many years, endo-β-1,4-xylanases (EC 3.2.1.8) (referred to herein asxylanases) have been used for the modification of complex carbohydratesderived from plant cell wall material. It is well known in the art thatthe functionality of different xylanases (derived from different microorganisms or plants) differs enormously. Based on structural and geneticinformation, xylanases have been classified into different GlycosideHydrolase families (GH's) (Henrissat, 1991; Coutinho and Henrissat,1999). Until recently, all known and characterized xylanases werebelonging to the families GH10 or GH11. Recent work has identifiednumerous other types of xylanases belonging to the families GH5, GH7,GH8 and GH43 (Coutinho and Henrissat, 1999; Collins et al., 2005). Untilnow the GH11 family differs from all other GH's, being the only familysolely consisting of xylan specific xylanases. The structure of the GH11xylanases can be described as a β-Jelly roll structure (see FIG. 1,discussed herein).

U.S. Pat. No. 6,682,923 relates to xylanase activity proteins andnucleic acids.

Comprehensive studies characterising the functionality of xylanases havebeen done on well characterised and pure substrates (Kormelink et al.,1992). These studies show that different xylanases have differentspecific requirements with respect to substitution of the xylosebackbone of the arabinoxylan (AX). Some xylanases require threeun-substituted xylose residues to hydrolyse the xylose backbone; othersrequire only one or two. The reasons for these differences inspecificity are thought to be due to the three dimensional structurewithin the catalytic domains, which in turn is dependent on the primarystructure of the xylanase, i.e. the amino acid sequence. However, thetranslation of these differences in the amino acid sequences intodifferences in the functionality of the xylanases, has up until now notbeen documented when the xylanase acts in a complex environment, such asplant material.

The xylanase substrates found in wheat (wheat flour), have traditionallybeen divided into two fractions: The water un-extractable AX (WU-AX) andthe water extractable AX (WE-AX). The WU-AX:WE-AX ratio is approx. 70:30in wheat flour. There have been numerous explanations as to why thereare two different fractions of AX. The older literature (D'Appolonia andMacArthur (1976) and Montgomery and Smith (1955)) describes quite highdifferences in the substitution degree between WE-AX and WU-AX. Thehighest degree of substitution was found in WE-AX. This was used toexplain why some of the AX was extractable. The high degree ofsubstitution made the polymer soluble, compared to a lower substitutiondegree, which would cause hydrogen bonding between polymers andconsequently precipitation.

The difference between the functionality of different xylanases has beenthought to be due to differences in xylanase specificity and therebytheir preference for the WU-AX or the WE-AX substrates.

However, more recent literature does not find the same huge differencesbetween the substitution degree of the WE-AX and the WU-AX. Hence otherparameters than the xylanases substrate specificity might be ofimportance. These parameters may be the xylanases preference for WE-AXversus WU-AX, determined by other means than classical substratespecificity. This parameter can be found described in literature assubstrate selectivity.

In some applications (e.g. bakery) it is desirable to produce highmolecular weight (HMW) soluble polymers from the WU-AX fraction. Suchpolymers have been correlated to a volume increase in bread making(Rouau, 1993; Rouau et al., 1994 and Courtin et al., 1999).

In other applications it is desirable to modify both the WU-AX andWE-AX, solubilising the WU-AX, making the molecular weight lower,reducing their hydrocolloid effect, produce arabinoxylanoligosaccharides, giving access to further degradation of other cellwall components (such as in crackers production, flour separation, feedapplication, Bio-ethanol production, Prebiotics, etc.).

All the above mentioned characteristics of xylanases used in variousapplications are directed to the xylanases performance and are of greatimportance to achieve the functionality needed. However, selection ofxylanases having the right characteristics for a certain application orengineering known xylanases to achieve it, often results in a lessefficient xylanase molecule, e.g., a molecule with low catalyticactivity (i.e., specific activity characterised by the moleculesunits/mg xylanase protein). Since these molecules are to be used incommercial applications it is therefore of great importance to have ashigh a catalytic activity as possible. Improvement of thischaracteristic will be of more and more importance to achieve commercialapplication of these enzymes in the future, due to the increased use ofagricultural by-products such as cereal bran or the use in cellulosicbio-ethanol production.

SUMMARY OF THE INVENTION

The present invention is predicated on the surprising finding that it ispossible—by modifying a polypeptide with xylanase activity at position110 compared to the B. subtilis xylanase polypeptide sequence shown asSEQ ID No. 1 to increase the bran solubilisation and/or xylanaseactivity of the enzyme.

Thus, it is has been shown by the inventors of the present inventionthat is possible to produce xylanase polypeptides having increasedxylanase activity and/or bran solubilisation. This will, for example,make it feasible to hydrolyse the hemicellulosic fraction during cerealprocessing related to cellulosic bioethanol production, or allow areduction in the amount of xylanase required in a number of applicationssuch as animal feed, starch liquefaction, bakery, flour separation(wetmilling), production of prebiotics, and paper and pulp production.

In a first aspect, the present invention relates to a polypeptide havingxylanase activity and comprising an amino acid sequence having at least88% identity with SEQ ID No. 1 or having at least 75% identity with anamino acid sequence selected from SEQ ID No. 2-22, and which polypeptidehas an amino acid modification in position 110, wherein said position110 is determined as the position corresponding to position 110 of B.subtilis xylanase sequence shown as SEQ ID No. 1 by alignment.

In a second aspect, the present invention relates to a polypeptidehaving xylanase activity and comprising an amino acid sequence having atleast 88% identity with SEQ ID No. 1 and which polypeptide has an aminoacid modification in position 110, wherein said position 110 isdetermined as the position corresponding to position 110 of B. subtilisxylanase sequence shown as SEQ ID No. 1 by alignment.

In a third aspect, the present invention relates to a polypeptide havingxylanase activity and comprising an amino acid sequence having at least75% identity with SEQ ID No. 2 and which polypeptide has an amino acidmodification in position 110, wherein said position 110 is determined asthe position corresponding to position 110 of B. subtilis xylanasesequence shown as SEQ ID No. 1 by alignment.

In a further aspect, the present invention relates to a polypeptidehaving xylanase activity and comprising an amino acid sequence having atleast 75% identity with SEQ ID No. 3 and which polypeptide has an aminoacid modification in position 110, wherein said position 110 isdetermined as the position corresponding to position 110 of B. subtilisxylanase sequence shown as SEQ ID No. 1 by alignment.

In a further aspect, the present invention relates to a method ofidentifying a polypeptide according to the invention, said methodcomprising:

(i) preparing a polypeptide having at least 88% identity with SEQ ID No.1 or having at least 75% identity with an amino acid sequence selectedfrom SEQ ID No. 2-22, and which polypeptide has an amino acidmodification in position 110, wherein said position 110 is determined asthe position corresponding to position 110 of B. subtilis xylanasesequence shown as SEQ ID No. 1 by alignment;(ii) comparing the bran solubilisation and/or xylanase activity of saidpolypeptide with the bran solubilisation and/or xylanase activity of theamino acid sequence selected among SEQ ID NOs: 1-22 with which is hasthe highest percentage of identity; and(iii) selecting the polypeptide if it has improved bran solubilisationand/or improved xylanase activity compared to the amino acid sequenceselected among SEQ ID NOs: 1-22 with which is has the highest percentageof identity.

In a further aspect, the present invention relates to a method ofpreparing a polypeptide according to the invention, said methodcomprising expressing a nucleotide sequence encoding said polypeptide;and optionally isolating and/or purifying the polypeptide afterexpression.

In some embodiments the polypeptide is prepared by modifying either apolypeptide amino acid sequence at position 110 or a codon that encodesan amino acid residue at position 110 in a nucleotide sequence encodinga polypeptide amino acid sequence, wherein position 110 is determinedwith reference to the B. subtilis xylanase sequence shown as SEQ ID No.1.

In a further aspect, the present invention relates to a nucleotidesequence encoding a polypeptide according to the invention.

In a further aspect, the present invention relates to a vectorcomprising the nucleotide sequence encoding a polypeptide according tothe invention.

In a further aspect, the present invention relates to a cell that hasbeen transformed with the nucleotide sequence encoding a polypeptideaccording to the invention or the vector comprising the nucleotidesequence encoding a polypeptide according to the invention.

In a further aspect, the present invention relates to a host organismthat has been transformed with the nucleotide sequence encoding apolypeptide according to the invention or the vector comprising thenucleotide sequence encoding a polypeptide according to the invention.

In a further aspect, the present invention relates to a compositioncomprising the polypeptide according to the invention.

In a further aspect, the present invention relates to a compositioncomprising a polypeptide identified according to the methods of theinvention

In a further aspect, the present invention relates to a compositioncomprising a polypeptide prepared according to the invention.

In a further aspect, the present invention relates to a compositioncomprising the nucleotide sequence encoding a polypeptide according tothe invention.

In a further aspect, the present invention relates to a compositioncomprising the vector comprising the nucleotide sequence encoding apolypeptide according to the invention

In a further aspect, the present invention relates to a compositioncomprising the cell that has been transformed with the nucleotidesequence encoding a polypeptide according to the invention.

In a further aspect, the present invention relates to a compositioncomprising the vector comprising the nucleotide sequence encoding apolypeptide according to the invention

In a further aspect, the present invention relates to a compositioncomprising the organism that has been transformed with the nucleotidesequence encoding a polypeptide according to the invention or the vectorcomprising the nucleotide sequence encoding a polypeptide according tothe invention admixed with a non toxic component.

In a further aspect, the present invention relates to a dough comprisingthe polypeptide according to the invention or a polypeptide identifiedaccording to the invention or a polypeptide prepared according to theinvention or the nucleotide sequence according to the invention or thevector according to the invention or the cell according to the inventionor the organism according to the invention admixed with a non toxiccomponent or a composition according to the invention.

In a further aspect, the present invention relates to a bakery productcomprising the polypeptide according to the invention or a polypeptideidentified according to the invention or a polypeptide preparedaccording to the invention or the nucleotide sequence according to theinvention or the vector according to the invention or the cell accordingto the invention or the organism according to the invention admixed witha non toxic component or a composition according to the invention or adough according to the invention.

In a further aspect, the present invention relates to an animal feedcomprising the polypeptide according to the invention or a polypeptideidentified according to the invention or a polypeptide preparedaccording to the invention or the nucleotide sequence according to theinvention or the vector according to the invention or the cell accordingto the invention or the organism according to the invention admixed witha non toxic component or a composition according to the invention.

In a further aspect, the present invention relates to a cleaningcomposition comprising xylanase. In some embodiments, the cleaningcompositions are laundry detergent compositions, while in otherembodiments the cleaning compositions are dishwashing detergents. Insome further embodiments, the dishwashing detergents are automaticdishwashing detergents. In some additional embodiments, thexylanase-containing cleaning compositions further comprise one or moreadditional enzymes. In some embodiments, the additional enzymes areselected from hemicellulases, cellulases, peroxidases, proteases,xylanases, lipases, phospholipases, esterases, cutinases, pectinases,pectate lyases, mannanases, keratinases, reductases, oxidases,phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases,pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase,chondroitinase, laccase, and amylases, or mixtures thereof. In someembodiments, a combination of enzymes finds use (i.e., a “cocktail”).

In a further aspect, the present invention relates to a method ofdegrading or modifying a plant cell wall which method comprisescontacting said plant cell wall with the polypeptide according to theinvention or a polypeptide identified according to the invention or apolypeptide prepared according to the invention or the nucleotidesequence according to the invention or the vector according to theinvention or the cell according to the invention or the organismaccording to the invention admixed with a non toxic component or acomposition according to the invention.

In a further aspect, the present invention relates to a method ofprocessing a plant material which method comprises contacting said plantmaterial with the polypeptide according to any one of the invention or apolypeptide identified according to the invention or a polypeptideprepared according to the invention or the nucleotide sequence accordingto the invention or the vector according to the invention or the cellaccording to the invention or the organism according to the inventionadmixed with a non toxic component or a composition according to theinvention.

In a further aspect, the present invention relates to the use of thepolypeptide according to the invention or a polypeptide identifiedaccording to the invention or a polypeptide prepared according to theinvention or the nucleotide sequence according to the invention or thevector according to the invention or the cell according to the inventionor the organism according to the invention admixed with a non toxiccomponent or a composition according to the invention in a method ofmodifying plant materials.

In a further aspect, the present invention relates to the use of thepolypeptide according to the invention or a polypeptide identifiedaccording to the invention or a polypeptide prepared according to theinvention or the nucleotide sequence according to the invention or thevector according to the invention or the cell according to the inventionor the organism according to the invention admixed with a non toxiccomponent or a composition according to the invention in any one or moreof: baking, processing cereals, starch liquefaction, production ofBio-ethanol from cellulosic material, animal feed, in processing wood,enhancing the bleaching of wood pulp.

In a further aspect, the present invention relates to a polypeptide orfragment thereof substantially as hereinbefore described with referenceto the Examples and drawings.

In a further aspect, the present invention relates to a methodsubstantially as hereinbefore described with reference to the Examplesand drawings.

In a further aspect, the present invention relates to a compositionsubstantially as hereinbefore described with reference to the Examplesand drawings.

In a further aspect, the present invention relates to the usesubstantially as hereinbefore described with reference to the Examplesand drawings.

LEGENDS TO THE FIGURE

Reference shall be made herein to the following Figure.

FIG. 1 shows the Bacillus subtilis XynA variant xylanase (T110A)(black), the Trichoderma reesei Xyn2 variant xylanase (T120A) (darkgrey) and the Thermomyces lanuginosus XynA variant xylanase (T120A)(light grey) superimposed. The residues mutated, T110 and T120respectively are highlighted.

FIG. 2 shows a multiple sequence alignment of SEQ ID NO:1-22 in theAlignX program (part of the vectorNTI suite) with default parameters formultiple alignment (Gap opening penalty: 10 og Gap extension penalty0.05). Numbers on the left of the sequence represent the SEQ ID NOs.

DETAILED DISCLOSURE OF THE INVENTION

Xylanase enzymes have been reported from nearly 100 different organisms,including plants, fungi and bacteria. The xylanase enzymes areclassified into several of the more than 40 families of glycosylhydrolase enzymes. The glycosyl hydrolase enzymes, which includexylanases, mannanases, amylases, β-glucanases, cellulases, and othercarbohydrases, are classified based on such properties as the sequenceof amino acids, the three dimensional structure and the geometry of thecatalytic site (Gilkes, et al., 1991, Microbiol. Reviews 55: 303-315).

In one aspect, the present invention relates to a polypeptide havingxylanase activity and comprising at least three, such as five, six,seven, eight, nine or ten amino acid substitutions relative to any oneamino acid sequence of SEQ ID NO:1-22, and which polypeptide has anamino acid substitution in position 110, wherein said position 110 isdetermined as the position corresponding to position 110 of B. subtilisxylanase sequence shown as SEQ ID No. 1 by alignment.

In one aspect, the present invention relates to a polypeptide havingxylanase activity and comprising an amino acid sequence, said amino acidsequence having at least 88% identity with SEQ ID No. 1 or having atleast 75% identity with an amino acid sequence selected from 2-22 andwhich polypeptide has an amino acid modification in position 110,wherein said position 110 is determined as the position corresponding toposition 110 of B. subtilis xylanase sequence shown as SEQ ID No. 1 byalignment.

The position of a particular amino acid within a polypeptide accordingto the present invention is determined by alignment of the amino acidsequence of said polypeptide with SEQ ID No. 1 using the a standardsequence alignment tool such as a by alignment of two sequences usingthe Smith-Waterman algorithm, or with the CLUSTALW2 algorithms, whereinthe sequences are said to be aligned when the alignment score ishighest. Alignment scores may be calculated according to the methodsdescribed by Wilbur, W. J. and Lipman, D. J. (1983) Rapid similaritysearches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci.USA, 80: 726-730. Preferably default parameters are used in theClustalW2 (1.82) algorithm: Protein Gap Open Penalty=10.0; Protein GapExtension Penalty=0.2; Protein matrix=Gonnet; Protein/DNA ENDGAP=−1;Protein/DNA GAPDIST=4.

Preferably a position of a particular amino acid within a polypeptideaccording to the present invention is determined by alignment of theamino acid sequence of the polypeptide with SEQ ID No. 1 using theAlignX program (part of the vectorNTI suite) with default parameters formultiple alignment (Gap opening penalty: 10 og Gap extension penalty0.05). For some embodiments according to the present invention,alignment may be made by using FIG. 2 as described herein.

In a further aspect, the present invention relates to a polypeptidehaving xylanase activity and having an amino acid sequence having atleast 88% identity with SEQ ID No. 1 and which polypeptide has an aminoacid modification in position 110, wherein said position 110 isdetermined as the position corresponding to position 110 of B. subtilisxylanase sequence shown as SEQ ID No. 1 by alignment.

In a further aspect, the present invention relates to a polypeptidehaving xylanase activity and having at least 75% identity with SEQ IDNo. 2 and which polypeptide has an amino acid modification in position110, wherein said position 110 is determined as the positioncorresponding to position 110 of B. subtilis xylanase sequence shown asSEQ ID No. 1 by alignment.

In a further aspect, the present invention relates to a polypeptidehaving xylanase activity and having at least 75% identity with SEQ IDNo. 3 and which polypeptide has an amino acid modification in position110, wherein said position 110 is determined as the positioncorresponding to position 110 of B. subtilis xylanase sequence shown asSEQ ID No. 1 by alignment.

In a further aspect, the present invention relates to a polypeptidehaving xylanase activity and having at least 75% identity with SEQ IDNo. 4 and which polypeptide has an amino acid modification in position110, wherein said position 110 is determined as the positioncorresponding to position 110 of B. subtilis xylanase sequence shown asSEQ ID No. 1 by alignment.

In a further aspect, the present invention relates to a polypeptidehaving xylanase activity and comprising an amino acid sequence having atleast 75% identity with SEQ ID No. 1, and which polypeptide has an aminoacid substitution in position 110 to any one different amino acidresidue selected from the group consisting of: glutamic acid,tryptophan, alanine and cysteine, wherein said position 110 isdetermined as the position corresponding to position 110 of B. subtilisxylanase sequence shown as SEQ ID No. 1 by alignment.

Unless otherwise stated the term “Sequence identity” for amino acids asused herein refers to the sequence identity calculated as(n_(ref)−n_(dif))·100/n_(ref), wherein n_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinn_(ref) is the number of residues in one of the sequences. Hence, theamino acid sequence ASTDYWQNWT will have a sequence identity of 80% withthe sequence ASTGYWQAWT (n_(dif)=2 and n_(ref)=10).

In some embodiments the sequence identity is determined by conventionalmethods, e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thesearch for similarity method of Pearson & Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, using the CLUSTAL W algorithm of Thompson etal., 1994, Nucleic Acids Res 22:467380, by computerized implementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group). The BLAST algorithm(Altschul et al., 1990, Mol. Biol. 215:403-10) for which software may beobtained through the National Center for Biotechnology Informationwww.ncbi.nlm.nih.gov/) may also be used. When using any of theaforementioned algorithms, the default parameters for “Window” length,gap penalty, etc., are used.

The term “modification” as used herein means any chemical modificationto any one amino acid or to the amino acid sequence of the polypeptideselected from SEQ ID NO: 1-22, as well as genetic manipulation of theDNA encoding that polypeptide. The modification can be substitutions,deletions and/or insertions of one or more amino acids as well asreplacements of one or more amino acid side chains. In some embodiments,the polypeptides have xylanase activity only have amino acidsubstitutions relative to SEQ ID No.1-22.

It is to be understood that “modification” in a given polypeptide isrelative to the polypeptide selected from SEQ ID NO: 1-22 with thehighest percentage sequence identity to this given polypeptide.

The terminology for amino acid substitutions used in this description isas follows. The first letter represents the amino acid naturally presentat a position of a particular sequence. The following number representsthe position relative to SEQ ID No. 1. The second letter represents thedifferent amino acid substituting for the natural amino acid. An exampleis D11F/R122D/T110A, wherein the aspartic acid at position 11 of SEQ IDNO:1 is replaced by a phenylalanine and the arginine at position 122 ofSEQ ID NO:1 is replaced by an aspartic acid, and the threonine atposition 110 is replaced by an alanine, all three mutations being in thesame polypeptide having xylanase activity.

Apart from the amino acid modifications in the polypeptides withxylanase activity according to the invention, the polypeptides may havefurther amino acid modifications of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala to Ser, Val to Ile, Asp to Glu, Thrto Ser, Ala to Gly, Ala to Thr, Ser to Asn, Ala to Val, Ser to Gly, Tyrto Phe, Ala to Pro, Lys to Arg, Asp to Asn, Leu to Ile, Leu to Val, Alato Glu, and Asp to Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-/V-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

The term “host organism”, as used herein, includes any cell type whichis susceptible to transformation, transfection, transduction, and thelike with a nucleic acid construct or expression vector comprising apolynucleotide encoding the polypeptides of the present invention.

For the present purposes, a xylanase means a protein or a polypeptidehaving xylanase activity.

The phrase “a polypeptide having xylanase activity” as used hereinrefers to any protein or polypeptide that has activity in a xylanaseassay such as described herein.

Xylanase activity can be measured using any assay, in which a substrateis employed that includes 1,4-beta-D-xylosidic endo-linkages in xylans.The pH and the temperature used in the assay are to be adapted to thexylanase in question. Examples of suitable pH values are 4, 5, 6, 7, 8,9, 10 or 11. Examples of suitable temperatures are 30, 35, 37, 40, 45,50, 55, 60, 65, 70 or 80° C. Different types of substrates are availablefor the determination of xylanase activity e.g. Xylazyme tablets(crosslinked, dyed xylan substrate, Megazyme, Bray, Ireland).

Preferably, xylanase activity is measured using the following assay.

Xylanase Assay (Endo-β-1,4-Xylanase Activity)

Samples were diluted in citric acid (0.1 M)—di-sodium-hydrogen phosphate(0.2 M) buffer, pH 5.0, to obtain approx. OD₅₉₀=0.7 in this assay. Threedifferent dilutions of the sample were pre-incubated for 5 minutes at40° C. At time=5 minutes, 1 Xylazyme tablet (crosslinked, dyed xylansubstrate, Megazyme, Bray, Ireland) was added to the enzyme solution ina reaction volume of 1 ml. At time=15 minutes the reaction wasterminated by adding 10 ml of 2% TRIS/NaOH, pH 12. Blanks were preparedusing 1000 μl buffer instead of enzyme solution. The reaction mixturewas centrifuged (1500×g, 10 minutes, 20° C.) and the OD of thesupernatant was measured at 590 nm. One xylanase unit (XU) is defined asthe xylanase activity increasing OD₅₉₀ with 0.025 per minute.

The substrate (cross-linked and dyed arabinoxylan extracted from wheat)used in the above assay is a good approximate to the correspondingsubstrate in commercial applications.

Enzymes can furthermore be classified on the basis of the handbookEnzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site atthe internet: www.expasy.ch/enzyme/. ENZYME is a repository ofinformation relative to the nomenclature of enzymes. It is primarilybased on the recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUB-MB) andit describes each type of characterized enzyme for which an EC (EnzymeCommission) number has been provided (Bairoch A. The ENZYME database,2000, Nucleic Acids Res 28:304-305). This IUB-MB Enzyme nomenclature isbased on their substrate specificity and occasionally on their molecularmechanism; such a classification does not reflect the structuralfeatures of these enzymes.

In one aspect of the invention, the xylanase is an enzyme classified asEC 3.2.1.8. The official name is endo-1,4-beta-xylanase. The systematicname is 1,4-beta-D-xylan xylanohydrolase. Other names may be used, suchas endo-(1-4)-beta-xylanase; (1-4)-beta-xylan 4-xylanohydrolase;endo-1,4-xylanase; xylanase; beta-1,4-xylanase; endo-1,4-xylanase;endo-beta-1,4-xylanase; endo-1,4-beta-D-xylanase; 1,4-beta-xylanxylanohydrolase; beta-xylanase; beta-1,4-xylan xylanohydrolase;endo-1,4-beta-xylanase; beta-D-xylanase. The reaction catalyzed is theendohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, xylanase, galactanase, mannanase, dextranase andalpha-galactosidase, in families based on amino acid sequencesimilarities has been proposed a few years ago. They currently fall into90 different families: See the CAZy(ModO) internet site (Coutinho, P. M.& Henrissat, B. (1999) Carbohydrate-Active Enzymes server at:afmb.cnrs-mrs.fr/˜cazy/CAZY/index.html (corresponding papers: Coutinho,P. M. & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrateddatabase approach. In “Recent Advances in Carbohydrate Bioengineering”,HJ. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., The RoyalSociety of Chemistry, Cambridge, pp. 3-12; Coutinho, P. M. & Henrissat,B. (1999) The modular structure of cellulases and othercarbohydrate-active enzymes: an integrated database approach. In“Genetics, Biochemistry and Ecology of Cellulose Degradation”, K.Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimuraeds., Uni Publishers Co., Tokyo, pp. 15-23).

In one aspect of the invention, the xylanase of the invention is axylanase of Glycoside Hydrolyase (GH) Family 11. The term “of GlycosideHydrolyase (GH) Family 11” means that the xylanase in question is or canbe classified in the GH family 11.

It is to be understood that protein similarity searches (likeProteinBlast at e.g. toolkit.tuebingen.mpg.de/prot_blast) may notnecessarily determine whether an unknown sequence actually falls underthe term of a GH11 xylanase family member. Proteins sequences foundusing a blast search might have relatively high identity/homology andstill not be actual xylanases, and furthermore, not be xylanasesbelonging to GH11. Alternatively, protein sequences may have arelatively low primary amino acid sequence identity and still be a GH11xylanase family member. To determine whether an unknown protein sequenceactually is a xylanase protein within the GH11 family, the evaluationwill have to be done, not only on sequence similarity, but also on3D—structure similarity, since the classification within GH-familiesrely on the 3D fold. A software that will predict the 3D fold of anunknown protein sequence is HHpred (toolkit.tuebingen.mpg.de/hhpred).The power of this software for protein structure prediction relies onidentifying homologous sequences with known structure to be used astemplate. This works so well because structures diverge much more slowlythan primary sequences. Proteins of the same family may have verysimilar structures even when their sequences have diverged beyondrecognition.

In practice, an unknown sequence can be pasted into the software(toolkit.tuebingen.mpg.de/hhpred) in FASTA format. Having done this, thesearch can be submitted. The output of the search will show a list ofsequences with known 3D structures. To confirm that the unknown sequenceindeed is a GH11 xylanase, GH11 xylanases should be found within thelist of homologues having a probability of >90. Not all proteinsidentified as homologues will be characterised as GH11 xylanases, butsome will. The latter proteins are proteins with a known structure andbiochemically characterisation identifying them as xylanases. The formerhave not been biochemically characterised as GH11 xylanases. Severalreferences describes this protocol such as Söding J. (2005) Proteinhomology detection by HMM-HMM comparison. Bioinformatics 21, 951-960(doi:10.1093/bioinformatics/bti125) and Söding J, Biegert A, and Lupas AN. (2005) The HHpred interactive server for protein homology detectionand structure prediction. Nucleic Acids Research 33, W244-W248 (WebServer issue) (doi:10.1093/nar/gki40).

According to the Cazy(ModO) site, Family 11 glycoside hydrolases can becharacterised as follows:

Known Activities: xylanase (EC 3.2.1.8)

Mechanism: Retaining

Catalytic Nucleophile/Base: Glu (experimental)

Catalytic Proton Donor: Glu (experimental)

3D Structure Status: Fold: β-jelly roll

Clan: GH-C

As used herein, “Clan C” refers to groupings of families which share acommon three-dimensional fold and identical catalytic machinery (see,for example, Henrissat, B. and Bairoch, A., (1996) Biochem. J., 316,695-696).

As used herein, “Family 11” refers to a family of enzymes as establishedby Henrissat and Bairoch (1993) Biochem J., 293, 781-788 (see, also,Henrissat and Davies (1997) Current Opinion in Structural Biol. 1997,&:637-644). Common features for family 11 members include high genetichomology, a size of about 20 kDa and a double displacement catalyticmechanism (see Tenkanen et al., 1992; Wakarchuk et al., 1994). Thestructure of the family 11 xylanases includes two large β-sheets made ofβ-strands and α-helices.

Family 11 xylanases include but are not limited to the following:Aspergillus niger XynA, Aspergillus kawachii XynC, Aspergillustubigensis XynA, Bacillus circulans XynA, Bacilluspunzilus XynA,Bacillus subtilis XynA, Neocalliniastix patriciarum XynA, Streptomyceslividans XynB, Streptomyces lividans XynC, Streptomyces therinoviolaceusXynII, Thermomonospora fusca XynA, Trichoderma harzianum Xyn,Tyichoderma reesei XynI, Trichoderma reesei XynII, TrichodermavirideXyn.

As used herein, “wild-type” refers to a sequence or a protein that isnative or naturally occurring.

In another particular embodiment, the xylanase of the invention isderived from a bacterial xylanase, such as from a bacterium of (i) thephylum of Firmicutes; (ii) the class of Bacilli; (iii) the order ofBacillales; (iv) the family of Paenibacillaceae; or (v) the genus ofPaenibacillus; such as from a bacterium of (vi) the species ofPaenibacillus pabuli, Paenibacillus polymyxa, or Paenibacillus sp.; suchas from (vii) strains of Paenibacillus pabuli, or Paenibacilluspolymyxa.

The expression “xylanase derived from a bacterial xylanase” as usedherein above includes any wild-type xylanase isolated from the bacteriumin question, as well as variants or fragments thereof which retainxylanase activity.

In a further particular embodiment the xylanase of the invention isderived from a fungal xylanase.

The above definition of “derived from” (in the context of bacterialxylanases) is applicable by analogy also to fungal xylanases.

Examples of fungal xylanases of family 11 glycoside hydrolase are thosewhich can be derived from the following fungal genera: Aspergillus,Aureobasidium, Emericella, Fusarium, Gaeumannomyces, Humicola,Lentinula, Magnaporthe, Neocallimastix, Nocardiopsis, Orpinomyces,Paecilomyces, Penicillium, Pichia, Schizophyllum, Talaromyces,Thermomyces, Trichoderma.

Fungal xylanases include yeast and filamentous fungal xylanases. In someembodiments, the xylanase is derived from a fungus of (i) the phylum ofAscomycota; (ii) the class of Pezizomycotina; (iii) the order ofEurotiomycetes; (iv) the sub-order of Eurotiales; (v) the family ofTrichocomaceae, such as the mitosporic Trichocomaceae; or from a fungusof (vi) the genus Aspergillus; such as from (vii) strains of Aspergillusniger. It will be understood that the definition of the aforementionedspecies includes both the perfect and imperfect states, and othertaxonomic equivalents e.g., anamorphs, regardless of the species name bywhich they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents.

Strains of the abovementioned bacteria and fungi are readily accessibleto the public in a number of culture collections, such as the AmericanType Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures(CBS), and Agricultural Research Service Patent Culture Collection,Northern Regional Research Center (NRRL).

Questions relating to taxonomy can be solved by consulting a taxonomydata base, such as the NCBI Taxonomy Browser which is available at thefollowing internet site:www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/. However, preferablyreference is to the following handbooks: Dictionary of the Fungi, 9thedition, edited by Kirk, P. M., P. F. Cannon, J. C. David & J. A.Stalpers, CAB Publishing, 2001; and Bergey's Manual of SystematicBacteriology, Second edition (2005).

The present invention relates to modification(s) at certain amino acidposition(s). These position(s) are listed with reference to the B.subtilis amino acid sequence shown as SEQ ID No. 1. In the presentinvention, the polypeptides with xylanase activity have a modificationat least in position 110 compared to the B. subtilis sequence shown asSEQ ID No. 1. Equivalent positions in other family 11 xylanases may befound by aligning other Family 11 xylanases with SEQ ID No. 1 anddetermining which amino acid aligns with the specific amino acid of SEQID No. 1 (e.g., see Example 5). Such alignment and use of one sequenceas a first reference is simply a matter of routine for one of ordinaryskill in the art.

In one aspect, a variant xylanase according to the invention has animproved bran solubilisation activity which is higher than what may beobtained by use of the corresponding wild-type xylanase, or any onexylanase comprising an amino acid sequence selected from SEQ ID No. 2-22as measured in a “bran solubilisation assay”.

In one aspect, the xylanase according to the invention has an improvedbran solubilisation activity as a result of the modification in position110.

Suitably, xylanase bran solubilising activity may be measured using thebran solubilising assay provided herein. Thus, polypeptides havingincreased xylanase activity and/or increased bran solubilising activitymay be provided. The requirement for specificity towards the WU-AX isincreasingly more and more important, since many applications are usingelevated concentration of cereal bran. The bread making industryincreases the bran concentration in many products, due to health andnutritional issues, the feed industry incorporates increasing amount ofbran material (fibre, Distillers Dried Grains with Solubles (DDGS)) dueto the use of cereal in Bioethanol production, for example. It istherefore advantageous to provide new xylanases with increasedspecificity, and hence efficacy in solubilising this bran material.

Bran Solubilisation Assay

Preferably, bran solubility is measured using the following assay.

A suspension of wheat bran in (0.1 M)—di-sodium-hydrogen phosphate (0.2M) buffer, pH 5.0 is prepared to an concentration of 1.33% bran (w/w).From this suspension, aliquots of 750 μl are transferred into eppendorphtubes under stirring. Each substrate tube is pre-heated for 5 minutes at40° C. Hereto, 250 μl enzyme solution is added, making the endconcentration of substrate 1%. Three dilutions (in duplicate) are madefrom each xylanases, with increasing enzyme concentration (0.33; 1.0 and3.0 μg xylanase/gram bran) to each time of determination (0, 30, 60 and240 minutes). As blank, a heat denaturated solution of the xylanase isused. The reaction is terminated to the given times, by transferring thetubes to a incubator set at 95° C. Heat denaturated samples are kept at4° C. until all enzyme reactions are terminated. When all enzymereactions are terminated, Eppendorph tubes are centrifuged to obtain aclear supernatant. The enzymes capability to solubilize bran isexpressed as the increase in reducing end groups as determined usingPAHBAH (Lever, 1972).

Since side activities, such as amylase activity, may interfere with theabove assay, bran solubilisation assay should only be carried out onpurified xylanase samples (see Ex. 2).

In one aspect, the xylanase according to the invention has a reducedsensitivity to a xylanase inhibitor as compared to any one wild typexylanase, or any one xylanase comprising an amino acid sequence selectedfrom SEQ ID No. 2-22.

In a further aspect, the polypeptide having xylanase activity accordingto the invention has a reduced sensitivity to a xylanase inhibitor as aresult of the modification in position 110 in combination with one ormore modification(s) at any one or more of amino acid positions: 11, 12,13, 34, 54, 77, 81, 99, 104, 113, 114, 118, 122, 141, 154, 159, 162,164, 166 and 175.

The inhibitor may be an inhibitor found naturally in plant tissues.

As used herein, the term “xylanase inhibitor” refers to a compound,typically a protein, whose role is to control the depolymerization ofcomplex carbohydrates, such as arabinoxylan, found in plant cell walls.These xylanase inhibitors are capable of reducing the activity ofnaturally occurring xylanase enzymes as well as those of fungal orbacterial origin. Although the presence of xylanase inhibitors have beenreported in cereal seeds (see for example McLauchlan et al 1999a; Rouauand Suget 1998).

McLauchlan et al (1999a) disclose the isolation and characterisation ofa protein from wheat that binds to and inhibits two family-11 xylanases.Likewise, WO 98/49278 demonstrates the effect of a wheat flour extracton the activity of a group of microbial xylanases all of which areclassified as family 11 xylanases. Debyser et al. (1999) also disclosethat endoxylanases from Aspergillus niger and Bacillus subtilis, whichare both members of the family 11 xylanases were inhibited by a wheatxylanase inhibitor called TAXI. McLauchlan et al (1999b) teach thatextracts from commercial flours such as wheat, barley, rye and maize arecapable of inhibiting both family 10 and 11 xylanases.

The xylanase inhibitor may be any suitable xylanase inhibitor. By way ofexample, the xylanase inhibitor may be the inhibitor described inWO-A-98/49278 and/or the xylanase inhibitor described by Rouau, X. andSurget, A. (1998), McLauchlan, R., et al. (1999) and/or the xylanaseinhibitor described in UK patent application number 9828599.2 (filed 23Dec. 1998), UK patent application number 9907805.7 (filed 6 Apr. 1999)and UK patent application number 9908645.6 (filed 15 Apr. 1999).

The inhibitors described in the prior art may also be used in assays todetermine the sensitivity of a variant polypeptide of the invention toxylanase inhibitors. They may also be used as described below tomodulate the functionality of a xylanase.

Xylanase Inhibitor Assay

Preferably, xylanase inhibition activity is measured using the followingassay.

100 μl inhibitor preparation (containing various concentrations ofxylanase inhibitor (for quantification see Xylanase inhibitorquantification below)), 250 μl xylanase solution (containing 12 XUxylanase/ml) and 650 μl buffer (0.1 M citric acid—0.2M di-sodiumhydrogen phosphate buffer, 1% BSA (Sigma-Aldrich, USA), pH 5.0) wasmixed. The mixture was thermostated for 5 minutes at 40.0° C. At time=5minutes one Xylazyme tablet was added. At time=15 minutes reaction wasterminated by adding 10 ml 2% TRIS/NaOH, pH 12. The reaction mixture wascentrifuged (1500×g, 10 minutes, 20° C.) and the supernatant measured at590 nm. The xylanase inhibition was calculated as residual activity in%, compared to the blank.

The endogenous endo-β-1,4-xylanase inhibitor used is obtainable fromwheat flour. The inhibitor is a di-peptide, having a MW of about 40 kDa(as measured by SDS-PAGE or mass spectrometry) and a pI of about 8 toabout 9.5.

Sequence analysis to date has revealed that the inhibitor has thesequence presented as SEQ ID No. 24 or is highly homologous thereto.

A method to quantify the inhibitor concentration in a give inhibitorpreparation can be found in Ex. 3

Blanks were prepared the same way, but substituting the inhibitorsolution with water.

The present invention also relates to nucleotide sequence encoding apolypeptide according to the invention comprising a nucleotide sequenceoperably linked to one or more control sequences that direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences. A polynucleotideencoding a polypeptide of the present invention may be manipulated in avariety of ways to provide for expression of the polypeptide.Manipulation of the polynucleotide's sequence prior to its insertioninto a vector may be desirable or necessary depending on the expressionvector. The techniques for modifying polynucleotide sequences utilizingrecombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene {amyM),Bacillus amyloliquefaciens alpha-amylase gene {amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase {glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae those phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Terminators for filamentous fungal host cells may be obtained from thegenes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Terminators for yeast host cells may be obtained from the genes forSaccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochromeC(CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphatedehydrogenase. Other useful terminators for yeast host cells aredescribed by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Leaders for filamentous fungal host cells may be obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Polyadenylation sequences for filamentous fungal host cells may beobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease, and Aspergillus n/geralpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprf), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tec, and tip operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding the polypeptide of the presentinvention, a promoter, and transcriptional and translational stopsignals. The various nucleic acids and control sequences describedherein may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleotide sequence encoding thepolypeptide at such sites. Alternatively, a nucleotide sequence encodingthe polypeptide of the present invention may be expressed by insertingthe nucleotide sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof. Insome embodiments the amdS and pyrG genes of Aspergillus nidulans orAspergillus oryzae and the bar gene of Streptomyces hygroscopicus areused in an Aspergillus cell.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome. For integration into the host cell genome, the vector may relyon the polynucleotide's sequence encoding the polypeptide or any otherelement of the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination. For autonomousreplication, the vector may further comprise an origin of replicationenabling the vector to replicate autonomously in the host cell inquestion. The origin of replication may be any plasmid replicatormediating autonomous replication which functions in a cell. The term“origin of replication” or “plasmid replicator” is defined herein as anucleotide sequence that enables a plasmid or vector to replicate invivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB1 10, pE194, pTA1060, and pAMβipermitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding the polypeptide of the present invention,which are advantageously used in the recombinant production of thepolypeptides. A vector comprising a polynucleotide encoding thepolypeptide of the present invention is introduced into a host cell sothat the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote. Useful unicellularmicroorganisms are bacterial cells such as gram positive bacteriaincluding, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens. Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans and Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In oneaspect, the bacterial host cell is a Bacillus lentus, Bacilluslicheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.In another aspect, the Bacillus cell is an alkalophilic Bacillus. Theintroduction of a vector into a bacterial host cell may, for instance,be effected by protoplast transformation (see, e.g., Chang and Cohen,1979, Molecular General Genetics 168: 111-115), using competent cells(see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThome, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In one aspect, the host cell is a fungal cell. “Fungi” as used hereinincludes the phyla Ascomycota, Basidiomycota, Chytridiomycota, andZygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby'sDictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra). In another aspect, the fungal hostcell is a yeast cell. “Yeast” as used herein includes ascosporogenousyeast (Endomycetales), basidiosporogenous yeast, and yeast belonging tothe Fungi Imperfecti (Blastomycetes). Since the classification of yeastmay change in the future, for the purposes of this invention, yeastshall be defined as described in Biology and Activities of Yeast(Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App.Bacteriol. Symposium Series No. 9, 1980).

In an even further aspect, the yeast host cell is a Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiacell.

In one particular aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another aspect, the yeasthost cell is a Kluyveromyces lactis cell. In another aspect, the yeasthost cell is a Yarrowia lipolytica cell.

In another aspect, the fungal host cell is a filamentous fungal cell.“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).The filamentous fungi are generally characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In another aspect, the filamentous fungal host cell is an Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In another aspect, the filamentous fungal host cell is an Aspergillusawamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzaecell. In another aspect, the filamentous fungal host cell is a Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In anotheraspect, the filamentous fungal host cell is a Bjerkandera adusta,Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinuscinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa,Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotuseryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell. Fungalcells may be transformed by a process involving protoplast formation,transformation of the protoplasts, and regeneration of the cell wall ina manner known per se. Suitable procedures for transformation ofAspergillus and Trichoderma host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75; 1920.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. Preferably, the cell isof the genus Aspergillus and more preferably Aspergillus fumigatus.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate.

For example, an enzyme assay may be used to determine the activity ofthe polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The polypeptides of the present invention may be purifiedby a variety of procedures known in the art including, but not limitedto, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

In one aspect, the amino acid modification in position 110 is an aminoacid substitution.

In one aspect, the amino acid modification in position 110 is an aminoacid deletion.

In one aspect, the amino acid modification in position 110 is an aminoacid insertion.

In some embodiments, the sequence identity is measured relative to SEQID No. 1, wherein the amino acid sequence according to SEQ ID No. 1further comprises a signal peptide sequence, such as its natural signalpeptide sequence.

In some embodiments, the polypeptide according to the invention has atleast 90, 92 or 95% identity with SEQ ID No. 1.

In some embodiments, the polypeptide according to the invention has atleast 76, 78, 80, 85, 90, 95, 98 or 95% identity with the sequence withwhich is has the highest percentage of identity selected from SEQ ID No.2-22.

In some embodiments, the polypeptide according to the invention has atleast 76, 78, 80, 85, 90, 95, 98 or 95% identity with SEQ ID No. 2.

In some embodiments, the polypeptide according to the invention has atleast 76, 78, 80, 85, 90, 95, 98 or 95% identity with SEQ ID No. 3.

In some embodiments, the polypeptide according to the invention has a3-jelly roll fold.

In some embodiments according to the invention the amino acidmodification in position 110 is an amino acid substitution.

In some embodiments according to the invention the amino acidmodification in position 110 is a substitution to any one differentamino acid residue selected from the group consisting of: alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, tryptophan, tyrosine and valine.

In some embodiments according to the invention the amino acidmodification in position 110 is a substitution to any one differentamino acid residue selected from the group consisting of: alanine,arginine, asparagine, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine and valine.

In some embodiments according to the invention the amino acidmodification in position 110 is a substitution to any one differentamino acid residue selected from the group consisting of: glutamic acid,tryptophan, alanine and cysteine.

In some embodiments according to the invention the amino acidmodification in position 110 is a substitution to alanine.

In some embodiments, the polypeptide according to the invention has atotal number of amino acids of less than 250, such as less than 240,such as less than 230, such as less than 220, such as less than 210,such as less than 200 amino acids, such as in the range of 160 to 240,such as in the range of 160 to 220 amino acids.

In some embodiments, the polypeptide according to the inventioncomprises one or more modification(s) at any one or more of amino acidpositions: 11, 12, 13, 34, 54, 77, 81, 99, 104, 113, 114, 118, 122, 141,154, 159, 162, 164, 166 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises one or more amino acid substitutions selected from the groupconsisting of: 11F, 12F, 54Q, 54W, 122D, 113A, 13Y, 113D, 175L, 122F,34K, 99Y, 104W, 141Q, 154R, 159D, 175K, 81I, 166F, 162E, 162D, 164F,114D, 114Y, 114F, 118V, 175K, 77L, 77M, 77S, 77V, and 77Y, theposition(s) being determined as the corresponding position of B.subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises one or more amino acid substitutions selected from the groupconsisting of: D11F, G12F, N54Q, R122D, Y113A, G13Y, Y113D, N141Q,Q175L, R122F, G34K, K99Y, T104W, K154R, N159D, Q175K, V81I, Y166F,S162E, S162D, W164F, N114D, N114Y, N114F, I118V, I77L, I77M, I77S, I77V,and I77Y, the position(s) being determined as the corresponding positionof B. subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises one or more modification(s) at any one or more of amino acidpositions: 13, 99, 104, 113, 122, 154, 159 and 175, the position(s)being determined as the corresponding position of B. subtilis amino acidsequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises substitution(s) at the amino acid positions: 13, 99, 104, 113,122, 154, 159 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the invention furthercomprises one or more modification(s) at any one or more of amino acidpositions: 114 and 166, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the invention furthercomprises one or more substitution(s) at any one or more of amino acidpositions: 114 and 166, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises substitution(s) in at least at four of the following aminoacid positions: 13, 99, 104, 113, 114, 122, 154, 159, 166, and 175, theposition(s) being determined as the corresponding position of B.subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises substitution(s) at the amino acid positions: 13, 99, 104, 113,114, 122, 154, 159 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises substitution(s) at the amino acid positions: 13, 99, 104, 113,122, 154, 159, 166 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises substitution(s) at the amino acid positions: 13, 99, 104, 113,122, 154, 159, and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises one or more amino acid substitutions selected from the groupconsisting of: 13Y, 99Y, 104W, 110A, 113D, 114D, 114F, 122F, 154R, 159D,166F, 175K, and 175L, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

In some embodiments, the polypeptide according to the invention has atleast five, six, seven, eight, nine or ten amino acid substitutionscompared to the sequence selected among SEQ ID No. 1-22 with which ithas the highest identity.

In some embodiments, the polypeptide according to the invention has atleast nine or ten amino acid substitutions.

In some embodiments, the polypeptide according to the inventioncomprises one or more amino acid substitutions selected from the groupconsisting of:

-   a. D11F, R122D and T110A;-   b. D11F, R122D, T110A and Y113A;-   c. G13Y, T110A, Y113D, R122D and Q175L;-   d. G13Y, T110A, Y113D, R122F and Q175L;-   e. G13Y, G34K, T110A, Y113D, R122D and Q175L;-   f. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175K,-   g. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D, Q175 and    V81I;-   h. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D, Y166F and    Q175L;-   i. G13Y, T110A, Y113D, R122D, K154R, N159D and Q175L;-   j. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and Q175L;-   k. G13Y, T110A, Y113D, R122D, S162E and Q175L;-   l. G13Y, T110A, Y113D, R122D, S162D and Q175L;-   m. G13Y, T110A, Y113D, R122D, W164F and Q175L;-   n. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175L;-   o. G13Y, K99Y, T104W, T110A, Y113D, N114Y, R122F, K154R, N159D and    Q175L;-   p. G13Y, K99Y, T104W, T110A, Y113D, N114F, R122F, K154R, N159D and    Q175L;-   q. G13Y, K99Y, T104W, T110A, Y113D, I118V, R122F, K154R, N159D and    Q175L;-   r. G13Y, K99Y, T104W, T110A, Y113D, N114Y, R122F, K154R, N159D and    Q175K;-   s. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175K;-   t. G13Y, I77L, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   u. G13Y, I77M, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   v. G13Y, I77S, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   w. G13Y, I77V, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   x. G13Y, I77Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L-   y. G13Y, K99Y, T104W, T110A, Y113D, R122F, N141Q, K154R, N159D,    Q175L;-   z. G13Y, N54Q, K99Y, T104W, T110A, Y113D, R122F, N141Q, K154R,    N159D, Q175,-   aa. G13Y, N54W, K99Y, T104W, T110A, Y113D, R122F, 141Q, K154R,    N159D, 175L;-   bb. G13Y, N54Q, K99Y, T104W, T110A, Y113D, N114F, R122F, K154R,    N159D, Q175L;-   cc. G13Y, K99Y, T104W, T110A, Y113D, N114F, R122F, 141Q, K154R,    N159D, Q175L; and-   dd. G13Y, 54Q, K99Y, T104W, T110A, Y113D, N114F, R122F, 141Q, K154R,    N159D, Q175L;    the position(s) being determined as the corresponding position of B.    subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the inventioncomprises one or more amino acid substitutions selected from the groupconsisting of:

-   a. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175K;-   b. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D, Y166F and    Q175L;-   c. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and Q175L;-   d. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175L;-   e. G13Y, K99Y, T104W, T110A, Y113D, N114F, R122F, K154R, N159D and    Q175L;    the position(s) being determined as the corresponding position of B.    subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the invention has bransolubilisation activity.

In some embodiments, the polypeptide according to the invention is inisolated form.

The term “isolated” as used herein means that the polypeptide is atleast substantially free from at least one other component with whichthe sequence is naturally associated in nature.

In some embodiments according to the invention the amino acidmodification in position 110 is not T110D.

In some embodiments the polypeptide having xylanase activity does nothave an aspartic acid in position 110.

In some embodiments, the polypeptide according to the invention hasimproved xylanase activity compared to the B. subtilis amino acidsequence shown as SEQ ID No. 1 as measured in a xylanase activity assay.

In some embodiments, the polypeptide according to the invention hasimproved xylanase activity as a result of the modification in position110.

In some embodiments, the polypeptide according to the invention hasimproved bran solubilisation activity compared to the B. subtilis aminoacid sequence shown as SEQ ID No. 1 as measured in a bran solubilisationactivity assay.

In some embodiments, the polypeptide according to the invention hasimproved bran solubilisation activity as a result of the modification inposition 110.

In some embodiments, the polypeptide according to the invention hasreduced sensitivity to a xylanase inhibitor.

In some embodiments, the polypeptide according to the invention has anamino acid sequence comprising modifications at positions selected fromthe list consisting of:

-   -   a) 13/110/113/122/154/159/175;    -   b) 13/99/104/110/113/122/154/159/166/175;    -   c) 13/99/104/110/113/114/122/154/159/175;    -   d) 13/110/113/122/175;    -   e) 13/99/104/110/113/122/154/159/175;    -   f) 13/99/104/110/113/122/154/159/175;    -   g) 13/99/104/110/113/114/122/154/159/175;    -   h) 13/99/104/110/113/114/122/154/159/175;    -   i) 13/99/104/110/113/114/122/154/159/175;    -   j) 13/99/104/110/113/114/122/154/159/175;    -   k) 13/77/99/104/110/113/122/154/159/175;    -   l) 13/81/99/104/110/113/122/154/159/175;    -   m) 13/110/113/122/164/175;    -   n) 13/110/113/122/162/175;    -   o) 13/110/113/122/175;    -   p) 11/122/110/113;    -   q) 13/77/99/104/110/113/122/154/159/175;    -   r) 11/122/110;    -   s) 13/34/110/113/122/175;    -   t) 13/77/99/104/110/113/122/154/159/175;    -   u) 13/77/99/104/110/113/122/154/159/175;    -   v) 13/99/104/110/113/118/122/154/159/175;    -   w) 13/110/113/122/162/175;    -   x) 13/77/99/104/110/113/122/154/159/175;    -   y) 13/99/104/110/113/122/141/154/159/175;    -   z) 13/54/99/104/110/113/122/141/154/159/175;    -   aa) 13/54/99/104/110/113/122/141/154/159/175;    -   bb) 13/54/99/104/110/113/114/122/154/159/175;    -   cc) 13/99/104/110/113/114/122/141/154/159/175; and    -   dd) 13/54/99/104/110/113/114/122/141/154/159/175;        the position(s) being determined as the corresponding position        of subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the invention has anamino acid sequence comprising amino acid substitutions selected fromthe list consisting of:

-   -   a) 13Y/110A/113D/122D/154R/159D/175L;    -   b) 13Y/99Y/104W/110A/113D/122F/154R/159D/166F/175L;    -   c) 13Y/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   d) 13Y/110A/113D/122F/175L;    -   e) 13Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   f) 13Y/99Y/104W/110A/113D/122F/154R/159D/175K;    -   g) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175K;    -   h) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175L;    -   i) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175L;    -   j) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175K;    -   k) 13Y/77L/99Y/104W/110A/113D/122F/154R/159D/175L;    -   l) 13Y/81I/99Y/104W/110A/113D/122F/154R/159D/175L;    -   m) 13Y/110A/113D/122D/164F/175L;    -   n) 13Y/110A/113D/122D/162D/175L;    -   o) 13Y/110A/113D/122D/175L;    -   p) 11F/122D/110A/113A;    -   q) 13Y/77Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   r) 11F/122D/110A;    -   s) 13Y/34K/110A/113D/122D/175L;    -   t) 13Y/77V/99Y/104W/110A/113D/122F/154R/159D/175L;    -   u) 13Y/77M/99Y/104W/110A/113D/122F/154R/159D/175L;    -   v) 13Y/99Y/104W/110A/113D/118V/122F/154R/159D/175L;    -   w) 13Y/110A/113D/122D/162E/175L;    -   x) 13Y/77S/99Y/104W/110A/113D/122F/154R/159D/175L;    -   y) 13Y/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   z) 13Y/54Q/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   aa) 13Y/54W/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   bb) 13Y/54Q/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   cc) 13Y/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L; and    -   dd) 13Y/54Q/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L;        the position(s) being determined as the corresponding position        of subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the invention has anamino acid sequence of SEQ ID No. 1 comprising amino acid substitutionsselected from the list consisting of

-   -   a) G13Y/T110A/Y113D/R122D/K154R/N159D/Q175L;    -   b) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Y166F/Q175L;    -   c) G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   d) G13Y/T110A/Y113D/R122F/Q175L;    -   e) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   f) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175K;    -   g) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175K;    -   h) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175L;    -   i) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175L;    -   j) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175K;    -   k) G13Y/I77L/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   l) G13Y/V81I/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   m) G13Y/T110A/Y113D/R122D/W164F/Q175L;    -   n) G13Y/T110A/Y113D/R122D/S162D/Q175L;    -   o) G13Y/T110A/Y113D/R122D/Q175L;    -   p) D11F/R122D/T110A/Y113A;    -   q) G13Y/I77Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   r) D11F/R122D/T110A;    -   s) G13Y/G34K/T110A/Y113D/R122D/Q175L;    -   t) G13Y/I77V/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   u) G13Y/I77M/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   v) G13Y/K99Y/T104W/T110A/Y113D/1118V/R122F/K154R/N159D/Q175L;    -   w) G13Y/T110A/Y113D/R122D/S162E/Q175L; and    -   x) G13Y/I77S/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L    -   y) G13Y/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   z)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   aa)        G13Y/N54W/K99Y/T104W/T110A/Y113D/R122F/141Q/K154R/N159D/175L,    -   bb)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   cc)        G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L;        and    -   dd) G13Y/54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159        D/Q175L.

In some embodiments, the polypeptide according to the invention has anamino acid sequence, which consists of amino acid substitutions selectedfrom the list consisting of:

-   -   a) 13Y/110A/113D/122D/154R/159D/175L;    -   b) 13Y/99Y/104W/110A/113D/122F/154R/159D/166F/175L;    -   c) 13Y/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   d) 13Y/110A/113D/122F/175L;    -   e) 13Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   f) 13Y/99Y/104W/110A/113D/122F/154R/159D/175K;    -   g) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175K;    -   h) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175L;    -   i) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175L;    -   j) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175K;    -   k) 13Y/77L/99Y/104W/110A/113D/122F/154R/159D/175L;    -   l) 13Y/81I/99Y/104W/110A/113D/122F/154R/159D/175L;    -   m) 13Y/110A/113D/122D/164F/175L;    -   n) 13Y/110A/113D/122D/162D/175L;    -   o) 13Y/110A/113D/122D/175L;    -   p) 11F/122D/110A/113A;    -   q) 13Y/77Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   r) 11F/122D/110A;    -   s) 13Y/34K/110A/113D/122D/175L;    -   t) 13Y/77V/99Y/104W/110A/113D/122F/154R/159D/175L;    -   u) 13Y/77M/99Y/104W/110A/113D/122F/154R/159D/175L;    -   v) 13Y/99Y/104W/110A/113D/118V/122F/154R/159D/175L;    -   w) 13Y/110A/113D/122D/162E/175L;    -   x) 13Y/77S/99Y/104W/110A/113D/122F/154R/159D/175L;    -   y) 13Y/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   z) 13Y/54Q/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   aa) 13Y/54W/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   bb) 13Y/54Q/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   cc) 13Y/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L; and    -   dd) 13Y/54Q/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L,        the position(s) being determined as the corresponding position        of subtilis amino acid sequence shown as SEQ ID No. 1.

In some embodiments, the polypeptide according to the invention has anamino acid sequence of SEQ ID No. 1, which consists of amino acidsubstitutions selected from the list consisting of

-   -   a) G13Y/T110A/Y113D/R122D/K154R/N159D/Q175L;    -   b) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Y166F/Q175L;    -   c) G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   d) G13Y/T110A/Y113D/R122F/Q175L;    -   e) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   f) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175K;    -   g) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175K;    -   h) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175L;    -   i) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175L;    -   j) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175K;    -   k) G13Y/I77L/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   l) G13Y/V81I/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   m) G13Y/T110A/Y113D/R122D/W164F/Q175L;    -   n) G13Y/T110A/Y113D/R122D/S162D/Q175L;    -   o) G13Y/T110A/Y113D/R122D/Q175L;    -   p) D11F/R122D/T110A/Y113A;    -   q) G13Y/I77Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   r) D11F/R122D/T110A;    -   s) G13Y/G34K/T110A/Y113D/R122D/Q175L;    -   t) G13Y/I77V/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   u) G13Y/I77M/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   v) G13Y/K99Y/T104W/T110A/Y113D/1118V/R122F/K154R/N159D/Q175L;    -   w) G13Y/T110A/Y113D/R122D/S162E/Q175L; and    -   x) G13Y/I77S/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L    -   y) G13Y/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   z)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   aa)        G13Y/N54W/K99Y/T104W/T110A/Y113D/R122F/141Q/K154R/N159D/175L;    -   bb)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   cc)        G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L;        and    -   dd)        G13Y/54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L.

In some embodiments, the polypeptide according to the invention is notSEQ ID No. 25.

In some embodiments, the polypeptide according to the invention does nothave a sequence selected from the list consisting of:

SEQ ID No. 57 of International Patent Application WO0238746;

SEQ ID No. 62 of International Patent Application WO0238746;

SEQ ID No. 59 of International Patent Application WO0238746;

SEQ ID No. 53 of International Patent Application WO0238746;

SEQ ID No. 65 of International Patent Application WO0238746;

SEQ ID No. 56 of International Patent Application WO0238746;

SEQ ID No. 64 of International Patent Application WO0238746:

SEQ ID No. 52 of International Patent Application WO0238746;

SEQ ID No. 63 of International Patent Application WO0238746;

SEQ ID No. 61 of International Patent Application WO0238746;

SEQ ID No. 60 of International Patent Application WO0238746;

SEQ ID No. 58 of International Patent Application WO0238746;

SEQ ID No. 55 of International Patent Application WO0238746;

SEQ ID No. 12 of International Patent Application WO0238746;

SEQ ID No. 11 of International Patent Application WO0238746;

SEQ ID No. 21 of International Patent Application WO0068396; and

SEQ ID No. 22 of International Patent Application WO0068396.

In some embodiments of the present invention, the amino acid sequence isused for large scale applications.

Preferably the amino acid sequence is produced in a quantity of from 1 gper liter to about 100 g per liter of the total cell culture volumeafter cultivation of the host organism.

The present invention also relates to a composition comprising aminoacid sequences and/or nucleotide sequences encoding a xylanase asdescribed herein.

The composition of the present invention can lead to improved aroma,flavour, mildness, consistency, texture, body, mouth feel, firmness,viscosity, gel fracture, structure and/or organoleptic properties andnutrition of products for consumption containing said composition.Furthermore, the composition of the present invention can also be usedin combination with other components of products for consumption todeliver said improvements.

Although it is preferred that the composition of the present inventionis used to improve the aroma, flavour, mildness, consistency, texture,body, mouth feel, firmness, viscosity, gel fracture, structure,smoothness of the surface and/or organoleptic properties and nutritionof products for consumption containing said composition—the presentinvention also covers using the composition of the present invention asa component of pharmaceutical combinations with other components todeliver medical or physiological benefit to the consumer.

Accordingly, the composition of the present invention may be used incombination with other components.

Examples of other components include one or more of: thickeners, gellingagents, emulsifiers, binders, crystal modifiers, sweetners (includingartificial sweeteners), rheology modifiers, stabilisers, anti-oxidants,dyes, enzymes, carriers, vehicles, excipients, diluents, lubricatingagents, flavouring agents, colouring matter, suspending agents,disintegrants, granulation binders etc. These other components may benatural. These other components may be prepared by use of chemicaland/or enzymatic techniques.

As used herein the term “thickener or gelling agent” as used hereinrefers to a product that prevents separation by slowing or preventingthe movement of particles, either droplets of immiscible liquids, air orinsoluble solids. Thickening occurs when individual hydrated moleculescause an increase in viscosity, slowing the separation. Gelation occurswhen the hydrated molecules link to form a three-dimensional networkthat traps the particles, thereby immobilizing them.

The term “stabiliser” as used here is defined as an ingredient orcombination of ingredients that keeps a product (e.g. a food product)from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. afood product ingredient) that prevents the separation of emulsions.Emulsions are two immiscible substances, one present in droplet form,contained within the other. Emulsions can consist of oil-in-water, wherethe droplet or dispersed phase is oil and the continuous phase is water;or water-in-oil, where the water becomes the dispersed phase and thecontinuous phase is oil. Foams, which are gas-in-liquid, andsuspensions, which are solid-in-liquid, can also be stabilised throughthe use of emulsifiers. Aeration can occur in a three phase system whereair is entrapped by liquid oil then stabilised by agglomerated fatcrystals stabilised with an emulsifier. Emulsifiers have a polar groupwith an affinity for water (hydrophilic) and a non-polar group which isattracted to oil (lipophilic). They are absorbed at the interfaces ofthe two substances, providing an interfacial film acting to stabilisethe emulsion. The hydrophilic/lipophilic properties of emulsifiers areaffected by the structure of the molecule. These properties areidentified by the hydrophilic/lipophilic balance (HLB) value. Low HLBvalues indicate greater lipophilic tendencies which are used tostabilise water-in-oil emulsions. High HLB values are assigned tohydrophilic emulsifiers, typically used in oil-in-water emulsions. Thesevalues are derived from simple systems. Because foods often containother ingredients that affect the emulsification properties, the HLBvalues may not always be a reliable guide for emulsifier selection.

As used herein the term “binder” refers to an ingredient (e.g. a foodingredient) that binds the product together through a physical orchemical reaction. During “elation” for instance, water is absorbed,providing a binding effect. However, binders can absorb other liquids,such as oils, holding them within the product. In the context of thepresent invention binders would typically be used in solid orlow-moisture products for instance baking products: pastries, doughnuts,bread and others.

The term “crystal modifier” as used herein refers to an ingredient (e.g.a food ingredient) that affects the crystallisation of either fat orwater. Stabilisation of ice crystals is important for two reasons. Thefirst is directly related to the product stability from a separationstandpoint. The more freeze/thaw cycles a product encounters, the largerthe ice crystals become. These large crystals can break down productstructure, either naturally occurring, as in the case of cell walls, orthat which is created by “elation”. Because the water is no longer heldin place, the product may exhibit syneresis, or weeping, after thawing.

Secondly, in the case of a product which is consumed frozen, these largecrystals result in an undesirable, gritty mouth feel.

“Carriers” or “vehicles” mean materials suitable for compoundadministration and include any such material known in the art such as,for example, any liquid, gel, solvent, liquid diluent, solubilizer, orthe like, which is non-toxic and which does not interact with anycomponents of the composition in a deleterious manner.

Examples of nutritionally acceptable carriers include, for example,water, salt solutions, alcohol, silicone, waxes, petroleum jelly,vegetable oils, polyethylene glycols, propylene glycol, liposomes,sugars, gelatin, lactose, amylose, magnesium stearate, talc,surfactants, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Examples of excipients include one or more of: microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar andhigh molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferablycorn, potato or tapioca starch), sodium starch glycollate,croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of:polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesiumstearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propyleneglycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes).

As used herein the term “component suitable for animal or humanconsumption” means a compound which is or can be added to thecomposition of the present invention as a supplement which may be ofnutritional benefit, a fibre substitute or have a generally beneficialeffect to the consumer. The ingredients can be used in a wide variety ofproducts that require gelling, texturising, stabilising, suspending,film-forming and structuring, retention of juiciness, without addingunnecessary viscosity. Preferably, the ingredients will be able toimprove the shelf live and stability of the viable culture.

By way of example, the components may be prebiotics such as alginate,xanthan, pectin, locust bean gum (LBG), inulin, guar gum,galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),lactosucrose, soybean oligosaccharides, palatinose,isomalto-oligosaccharides, gluco-oligosaccharides andxylo-oligosaccharides.

The composition of the present invention may be used as—or in thepreparation of—a food. Here, the term “food” is used in a broadsense—and covers food for humans as well as food for animals (i.e. afeed). In a preferred aspect, the food is for human consumption.

The food may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

When used as—or in the preparation of—a food—such as functional food—thecomposition of the present invention may be used in conjunction with oneor more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

The composition of the present invention may be used as a foodingredient.

As used herein the term “food ingredient” includes a formulation whichis or can be added to functional foods or foodstuffs as a nutritionalsupplement and/or fiber supplement. The term food ingredient as usedhere also refers to formulations which can be used at low levels in awide variety of products that require gelling, texturising, stabilising,suspending, film-forming and structuring, retention of juiciness andimproved mouthfeel, without adding viscosity.

The food ingredient may be in the from of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The composition of the present invention may be—or may be added to—foodsupplements.

The composition of the present invention may be—or may be addedto—functional foods.

As used herein, the term “functional food” means food which is capableof providing not only a nutritional effect and/or a taste satisfaction,but is also capable of delivering a further beneficial effect toconsumer.

Accordingly, functional foods are ordinary foods that have components oringredients (such as those described herein) incorporated into them thatimpart to the food a specific functional—e.g. medical or physiologicalbenefit—other than a purely nutritional effect.

Although there is no legal definition of a functional food, most of theparties with an interest in this area agree that they are foods marketedas having specific health effects.

Some functional foods are nutraceuticals. Here, the term “nutraceutical”means a food which is capable of providing not only a nutritional effectand/or a taste satisfaction, but is also capable of delivering atherapeutic (or other beneficial) effect to the consumer. Nutraceuticalscross the traditional dividing lines between foods and medicine.

Surveys have suggested that consumers place the most emphasis onfunctional food claims relating to heart disease. Preventing cancer isanother aspect of nutrition which interests consumers a great deal, butinterestingly this is the area that consumers feel they can exert leastcontrol over. In fact, according to the World Health Organization, atleast 35% of cancer cases are diet-related. Furthermore claims relatingto osteoporosis, gut health and obesity effects are also key factorsthat are likely to incite functional food purchase and drive marketdevelopment.

The composition of the present invention can be used in the preparationof food products such as one or more of: jams, marmalades, jellies,dairy products (such as milk or cheese), meat products, poultryproducts, fish products and bakery products.

By way of example, the composition of the present invention can be usedas ingredients to soft drinks, a fruit juice or a beverage comprisingwhey protein, health teas, cocoa drinks, milk drinks and lactic acidbacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, waterices and desserts, confectionery, biscuits cakes and cake mixes, snackfoods, breakfast cereals, instant noodles and cup noodles, instant soupsand cup soups, balanced foods and drinks, sweeteners, texture improvedsnack bars, fibre bars, bake stable fruit fillings, care glaze,chocolate bakery filling, cheese cake flavoured filling, fruit flavouredcake filling, cake and doughnut icing, heat stable bakery filling,instant bakery filling creams, filing for cookies, ready-to-use bakeryfilling, reduced calorie filling, adult nutritional beverage, acidifiedsoy/juice beverage, aseptic/retorted chocolate drink, bar mixes,beverage powders, calcium fortified soy/plain and chocolate milk,calcium fortified coffee beverage.

A composition according to the present invention can further be used asan ingredient in food products such as American cheese sauce,anti-caking agent for grated & shredded cheese, chip dip, cream cheese,dry blended whip topping fat free sour cream, freeze/thaw dairy whippingcream, freeze/thaw stable whipped tipping, low fat & lite naturalcheddar cheese, low fat Swiss style yoghurt, aerated frozen desserts,and novelty bars, hard pack ice cream, label friendly, improvedeconomics & indulgence of hard pack ice cream, low fat ice cream: softserve, barbecue sauce, cheese dip sauce, cottage cheese dressing, drymix Alfredo sauce, mix cheese sauce, dry mix tomato sauce and others.

For certain aspects, preferably the foodstuff is a beverage.

For certain aspects, preferably the foodstuff is a bakery product—suchas bread, Danish pastry, biscuits or cookies.

The present invention also provides a method of preparing a food or afood ingredient, the method comprising xylanase produced by the processof the present invention or the composition according to the presentinvention with another food ingredient. The method for preparing or afood ingredient is also another aspect of the present invention.

In a general sense, a polypeptide having xylanase activity of theinvention may be used to solubilize and/or degrade insoluble plant cellwall material containing arabinoxylan, alter, for example reduce, theviscosity derived from the presence of hemicellulose or arabinoxylan ina solution or system comprising plant cell wall material. Typically saidplant cell wall materials will comprise one or more xylanase inhibitors.

Specifically, a polypeptide having xylanase activity of the inventionmay be used in processing plant materials for use as foodstuffs, such asanimal feed, in starch production, in baking, in production ofBio-ethanol from cellulosic material and in the processing of wood pulpto make paper.

A polypeptide having xylanase activity of the invention may be used toprocess plant materials such as cereals that are used in foodstuffsincluding animal feed. As used herein, the term “cereal” means any kindof grain used for food and/or any grass producing this grain such as butnot limited to any one of wheat, milled wheat, barley, maize, sorghum,rye, oats, triticale and rice or combinations thereof. In one preferredembodiment, the cereal is a wheat cereal.

The xylan in the food and/or feed supplement is modified by contactingthe xylan with the polypeptide having xylanase activity of the presentinvention.

As used herein, the term “contacting” includes but is not limited tospraying, coating, impregnating or layering the food and/or feedsupplement with the polypeptide having xylanase activity of the presentinvention.

In one embodiment, the food and/or feed supplement of the presentinvention may be prepared by mixing the polypeptide having xylanaseactivity directly with a food and/or feed supplement. By way of example,the polypeptide having xylanase activity may be contacted (for example,by spraying) onto a cereal-based food and/or feed supplement such asmilled wheat, maize or soya flour.

It is also possible to incorporate the polypeptide having xylanaseactivity it into a second (and different) food and/or feed or drinkingwater which is then added to the food and/or feed supplement of thepresent invention. Accordingly, it is not essential that the polypeptidehaving xylanase activity provided by the present invention isincorporated into the cereal-based food and/or feed supplement itself,although such incorporation forms a particularly preferred aspect of thepresent invention.

In one embodiment of the present invention, the food and/or feedsupplement may be combined with other food and/or feed components toproduce a cereal-based food and/or feed. Such other food and/or feedcomponents may include one or more other (preferably thermostable)enzyme supplements, vitamin food and/or feed supplements, mineral foodand/or feed supplements and amino acid food and/or feed supplements. Theresulting (combined) food and/or feed supplement comprising possiblyseveral different types of compounds can then be mixed in an appropriateamount with the other food and/or feed components such as cereal andprotein supplements to form a human food and/or an animal feed.

In one preferred embodiment, the food and/or feed supplement of thepresent invention can be prepared by mixing different enzymes having theappropriate activities to produce an enzyme mix. By way of example, acereal-based food and/or feed supplement formed from e.g. milled wheator maize may be contacted (e.g. by spraying) either simultaneously orsequentially with the xylanase enzyme and other enzymes havingappropriate activities. These enzymes may include but are not limited toany one or more of an amylase, a glucoamylase, a mannanase, agalactosidase, a phytase, a lipase, a phospholipase, a galactolipase, aglucanase, an-arabinofuranosidase, a ferulyol esterase, a pectinase, aprotease, a glucose oxidase, a hexose oxidase and a xylanase. Enzymeshaving the desired activities may for instance be mixed with thexylanase of the present invention either before contacting these enzymeswith a cereal-based food and/or feed supplement or alternatively suchenzymes may be contacted simultaneously or sequentially on such a cerealbased supplement. The food and/or feed supplement is then in turn mixedwith a cereal-based food and/or feed to prepare the final food and/orfeed. It is also possible to formulate the food and/or feed supplementas a solution of the individual enzyme activities and then mix thissolution with a food and/or feed material prior to processing the foodand/or feed supplement into pellets or as a mash.

The present invention provides the use of a polypeptide having xylanaseactivity of the invention in a process for preparing a foodstuff.Typical bakery (baked) products in accordance with the present inventioninclude bread—such as loaves, rolls, buns, pizza bases etc.—pretzels,tortillas, cakes, cookies, biscuits, crackers etc. The preparation offoodstuffs such as bakery products is well know in the art. Doughproduction, for example, is described in example 4. The use of apolypeptide having xylanase activity of the invention to alter thebaking performance is described in example 4.

A polypeptide having xylanase activity of the invention may also be usedin starch production from plant materials derived from cereals andtubers, such as potatoes.

A polypeptide having xylanase activity of the invention may also be usedin processing wood pulp, for example in the preparation of paper.

Processing of Cellulosic Material for Bio-Ethanol Production

A polypeptide having xylanase activity of the invention may also be usedin the hydrolysis of cellulosic plant material for production of sugarsfermentable to bio-ethanol.

In some particular embodiments the polypeptide having xylanase activityaccording to the invention has an optimal xylanase activity at doughprocessing temperatures, such as in the range of about 20 to about 40°C. In some embodiments the polypeptide having xylanase activityaccording to the invention are inactivated during a baking process.

In some alternative embodiments the polypeptide having xylanase activityaccording to the invention has increased thermostability and/ortemperature optimum as compared to the corresponding wild type enzyme toretain activity after heat treatment. Both characteristics are known topersons skilled in the art.

EXAMPLES Example 1 Site-Directed Mutagensis of Xylanases and Expression

Specific mutants of the Bacillus subtilis xylanase were obtained using aconstruct comprising the ribosome binding site from pET24a(ctagaaataattttgtttaactttaagaaggagatatacat) fused to the wild typexylanase gene without signal sequence (atggctagcacagactactggcaa - - -tggtaa) was transferred to the vector pCRBlunt (InVitrogen, Carlsbad,Calif., USA). This resulted in constitutive expression of xylanase inTOP10 cells (InVitrogen) after transformation with the constructedvector, provided that the orientation of the gene is in a “clockwise”direction. Site directed mutation in the gene was then obtained by theuse of the “QuickChange” mutagenesis kit (Stratagene, La Jolla, Calif.,USA) according to the manufacturers protocol. Mutants were verified bysequencing. Sufficient production of the verified mutants was obtainedby growing the transformed TOP10 cells in 1 L scale.

Example 2 Bran Solubilisation Studies of Xylanase Mutants

We used wheat bran as substrate to evaluate the specific activity of thexylanase variants since this is used in commercial applications.

Bran Substrate:

By means of example, bran could be wheat bran obtained from dry millingof wheat using a lab scale Chopin CD Auto Mill (Chopin Technologies,France), using the setting and conditions provided by the supplier, formilling wheat into wheat flour and bran. The obtained bran fraction maybe used as substrate in the bran solubilisation assay. In this Examplewheat was used as the cereal source.

Bran Solubilisation Assay:

A suspension of wheat bran in (0.1 M)—di-sodium-hydrogen phosphate (0.2M) buffer, pH 5.0 is prepared to an concentration of 1.33% bran (w/w).From this suspension, aliquots of 750 l are transferred into eppendorphtubes under stirring. Each substrate tube is pre-heated for 5 minutes at40° C. Hereto, 250 μl enzyme solution is added, making the endconcentration of substrate 1%. Three dilutions (in duplicate) are madefrom each xylanases, with increasing enzyme concentration (0.33; 1.0 and3.0 μg xylanase/gram bran) to each time of determination (0, 30, 60 and240 minutes). As blank, a heat denaturated solution of the xylanase isused. The reaction is terminated to the given times, by transferring thetubes to an incubator set at 95° C. Heat denaturated samples are kept at4° C. until all enzyme reactions are terminated. When all enzymereactions are terminated, Eppendorph tubes are centrifuged to obtain aclear supernatant. The enzymes capability to solubilize bran isexpressed as OD₄₁₀ increase, determined by the increase in reducing endgroups using PAHBAH reagens (Lever, 1972).

In short, reducing end groups are reacted with PAHBAH forming a coloredreaction product, which can be quantified at OD OD₄₁₀.

The above bran solubilisation assay is sensitive to side activity ofenzymes active on residual starch in the bran substrate.

Bacillus subtilis Xylanase Purification Protocol:

E. coli TOP10 cells having expressed the xylanase were harvested bycentrifugation (20 minutes, 3500×g, 20° C.) and resuspended in 50 mMTris, 2 mM EDTA, pH 7.4. Cells were opened by addition of 1 mg/mllysozyme (ICN Biomedicals, Costa Mesa, Calif., US, cat. No. 100831),stirring of the slurry for 2 hours at ambient temperature, freezing andthawing followed by sonication. pH was adjusted to 4.0 using 1M HClfollowed by centrifugation (20 minutes, 3500×g, 20° C.). The supernatantcontaining the xylanase was desalted using disposable PD-10 desaltingcolumns (Amersham Bioscience, Sweden) equilibrated in and eluted with 50mM sodium acetate, pH 4.5. The desalted sample was loaded onto a 10 mlSOURCE 15S column (Amersham Bioscience, Sweden) pre-equilibrated with 50mM sodium acetate, pH 4.5. The column was then washed with equilibrationbuffer and eluted with a linear NaCl gradient (50 mM sodium acetate,0-0.35M NaCl, pH 4.5). Fractions containing xylanase activity werepooled and used for further analysis.

Similar protocols may be adapted to non-Bacillus subtilis XynA derivedxylanase variants having a pI significantly different from Bacillussubtilis XynA

TABLE 1 Xylanases bran solubilising activity expressed as, maximumoptical density, slope index of xylanase mutants, relative opticaldensity and slope compared to the xylanase BS1 (the Bacillus subtilisenzyme shown as SEQ ID No. 1) and the xylanase BS3 (Bacillus subtilisvariant shown as SEQ ID No. 23) Slope OD/ Maximum Relative Relative Max.Relative Relative Max. Modifications made to SEQ ID No. 1 hr OD Slope toBS1 OD to BS1 Slope to BS3 OD to BS3 None (BS1) 0.23 0.69 100 100 140140 D11F/R122D (BS3) 0.16 0.49 72 72 100 100 D11F/R122D/T110A 0.25 0.75110 110 154 154 D11F/R122D/T110A/Y113A 0.27 0.80 117 117 163 163G13Y/T110A/Y113D/R122D/Q175L 0.27 0.82 119 119 167 167G13Y/T110A/Y113D/R122F/Q175L 0.37 1.12 164 164 229 229G13Y/G34K/T110A/Y113D/R122D/Q175L 0.25 0.74 108 108 150 150G13Y/K99Y/T104W/T110A/Y113D/R122F/ 0.37 1.11 162 162 226 226K154R/N159D/Q175K G13Y/K99Y/T104W/T110A/Y113D/R122F/ 0.29 0.87 128 128178 178 K154R/N159D/Q175/V81I G13Y/K99Y/T104W/T110A/Y113D/R122F/ 0.391.18 172 172 241 241 K154R/N159D/Y166F/Q175LG13Y/T110A/Y113D/R122D/K154R/ 0.40 1.19 173 173 242 242 N159D/Q175LG13Y/K99Y/T104W/T110A/Y113D/R122F/ 0.37 1.11 162 162 226 226K154R/N159D/Q175L G13Y/T110A/Y113D/R122D/S162E/Q175L 0.18 0.53 78 78 109109 G13Y/T110A/Y113D/R122D/S162D/Q175L 0.28 0.83 121 121 169 169G13Y/T110A/Y113D/R122D/W164F/Q175L 0.28 0.83 121 121 169 169G13Y/K99Y/T104W/T110A/Y113D/N114D/ 0.34 1.02 149 149 208 208R122F/K154R/N159D/Q175L G13Y/K99Y/T104W/T110A/Y113D/N114Y/ 0.35 1.04 152152 212 212 R122F/K154R/N159D/Q175L G13Y/K99Y/T104W/T110A/Y113D/N114F/0.38 1.15 169 169 235 235 R122F/K154R/N159D/Q175LG13Y/K99Y/T104W/T110A/Y113D/I118V/ 0.21 0.63 92 92 128 128R122F/K154R/N159D/Q175L G13Y/K99Y/T104W/T110A/Y113D/N114Y/ 0.33 0.98 144144 201 201 R122F/K154R/N159D/Q175K G13Y/K99Y/T104W/T110A/Y113D/N114D/0.35 1.05 153 153 214 214 R122F/K154R/N159D/Q175KG13Y/I77L/K99Y/T104W/T110A/Y113D/ 0.31 0.93 136 136 190 190R122F/K154R/N159D/Q175L G13Y/I77M/K99Y/T104W/T110A/Y113D/ 0.22 0.67 9898 137 137 R122F/K154R/N159D/Q175L G13Y/I77V/K99Y/T104W/T110A/Y113D/0.23 0.69 101 101 141 141 R122F/K154R/N159D/Q175LG13Y/I77Y/K99Y/T104W/T110A/Y113D/ 0.27 0.80 117 117 163 163R122F/K154R/N159D/Q175L

Example 3 Testing of Xylanase Activity and Relative Inhibition by CerealXylanase Inhibitors

The mutants of Example 2 were tested for xylanase activity and relativesensitivity to a xylanase inhibitor by the protocols presented below andin accordance with the following teachings.

Xylanase Assay (Endo-β-1,4-Xylanase Activity)

Samples were diluted in citric acid (0.1 M)—di-sodium-hydrogen phosphate(0.2 M) buffer, pH 5.0, to obtain approx. OD₅₉₀=0.7 in this assay. Threedifferent dilutions of the sample were pre-incubated for 5 minutes at40° C. At time=5 minutes, 1 Xylazyme tablet (crosslinked, dyed xylansubstrate, Megazyme, Bray, Ireland) was added to the enzyme solution ina reaction volume of 1 ml. At time=15 minutes the reaction wasterminated by adding 10 ml of 2% TRIS/NaOH, pH 12. Blanks were preparedusing 1000 μl buffer instead of enzyme solution. The reaction mixturewas centrifuged (1500×g, 10 minutes, 20° C.) and the OD of thesupernatant was measured at 590 nm. One xylanase unit (XU) is defined asthe xylanase activity increasing OD₅₉₀ with 0.025 per minute.

Specific Activity Determination:

Optical density at 280 nm of the purified samples was measured fordetermining xylanase protein concentration. A theoretically calculated,specific OD₂₈₀ (Gasteiger et al., 2003) of 0.25 units/mg×ml was used forthe specific activity calculation of the Bacillus subtilis XynA derivedvariants. Xylanase activity was determined as described above.

Xylanase Inhibitor Assay

100 μl inhibitor preparation (containing various concentrations ofxylanase inhibitor (for quantification see Xylanase inhibitorquantification below)), 250 μl xylanase solution (containing 12 XUxylanase/ml) and 650 μl buffer (0.1 M citric acid—0.2M di-sodiumhydrogen phosphate buffer, 1% BSA (Sigma-Aldrich, USA), pH 5.0) wasmixed. The mixture was thermostated for 5 minutes at 40.0° C. At time=5minutes one Xylazyme tablet (crosslinked, dyed xylan substrate,Megazyme, Bray, Ireland) was added. At time=15 minutes reaction wasterminated by adding 10 ml 2% TRIS/NaOH, pH 12. The reaction mixture wascentrifuged (1500×g, 10 minutes, 20° C.) and the supernatant measured at590 nm. The xylanase inhibition was calculated as residual activity in%, compared to the blank. Blanks were prepared the same way, butsubstituting the inhibitor solution with water.

Xylanase Inhibitor Quantification:

1 XIU (Xylanase Inhibitor Unit) is defined as the amount of inhibitorthat decreases 1 XU of the Bacillus subtilis XynA xylanase (Seq ID No 1)to 0.5 XU under the conditions described below.

250 μl xylanase solution containing 12 XU/ml, approx. 100 μl xylanaseinhibitor solution and McIlvaine buffer, pH 5, to reach a reactionvolume of 1000 μl is pre-incubated for 5 minutes at 40° C. At t=5minutes, 1 Xylazyme tablet is added to the reaction mixture. At t=15minutes the reaction is terminated, by addition of 10 ml 2% TRIS/NaOH,pH 12. The solution is filtered and the absorbance of the supernatant ismeasured at 590 nm. By choosing several different concentrations ofinhibitor in the above assay, it is possible to create a plot of ODversus inhibitor concentration. Using the slope (a) and intercept (b)from this plot and the concentration of the xylanase it is possible tocalculate the amount of XIU in a given inhibitor solution (equation 1).XIU=((b/2)/−a)/x  Equation 1X=Xylanase units (XU) in the assayInhibitor Preparation:

A crude inhibitor preparation (containing both TAXI and XIP, hereafterreferred to as inhibitor preparation) was prepared from 1 kg wheat(Triticum aestivum) flour. The inhibitor preparation was extracted fromthe flour using water in a 1:3 ratio (w/w) followed by centrifugation(3500×g, 20 minutes, 4° C.). The extract was kept at 65° C. for 40minutes, centrifuged (3500×g, 20 minutes, 4° C.) and desalted usingdisposable PD-10 desalting columns (Amersham Bioscience, Sweden)pre-equilibrated with 20 mM sodium phosphate buffer, pH 7. TAXIconcentration in the inhibitor preparation was determined by asdescribed above. The protocol for purification and quantification ofTAXI is described elsewhere (Sibbesen and Sørensen, 2001). By mean ofexample only, the TAXI in the preparation could be SEQ ID No. 24 or asequence having 90% identity thereto.

TABLE 2 Xylanase activity (XU/mg) and xylanase inhibitor mutantsindicated as residual xylanase activity at increasing inhibitorconcentrations (XIU/ml assay). Specific % Activity % Activity % ActivityModifications made Activity @ 5.6 @ 33.5 @ 50 to SEQ ID No. 1 XU/mgXIU/ml XIU/ml XIU/ml None (BS1) 23,000 29 D11F R122D (BS3) 8,400 100 10095 D11F/R122D/T110A 14,992 98 100 D11F/R122D/T110A/ 13,362 99 Y113AG13Y/T110A/Y113D/ 59,571 50 R122D/Q175L G13Y/T110A/Y113D/ 53,855 65R122F/Q175L G13Y/G34K/T110A/ 15,876 97 Y113D/R122D/ Q175LG13Y/K99Y/T104W/ 47,975 72 T110A/Y113D/R122F/ K154R/N159D/ Q175KG13Y/K99Y/T104W/ 41,791 46 T110A/Y113D/R122F/ K154R/N159D/Q175/ V81IG13Y/K99Y/T104W/ 53,331 68 T110A/Y113D/R122F/ K154R/N159D/Y166F/ Q175LG13Y/T110A/Y113D/ 54,924 32 R122D/K154R/N159D/ Q175L G13Y/K99Y/T104W/54,811 64 T110A/Y113D/R122F/ K154R/N159D/Q175L G13Y/T110A/Y113D/ 55,24944 R122D/S162E/Q175L G13Y/T110A/Y113D/ 52,735 40 R122D/S162D/Q175LG13Y/T110A/Y113D/ 51,884 29 R122D/W164F/Q175L G13Y/K99Y/T104W/ 47,445 79T110A/Y113D/N114D/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 46,263 78T110A/Y113D/N114Y/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 42,077 79T110A/Y113D/N114F/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 27,363 79T110A/Y113D/I118V/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 35,906 84T110A/Y113D/N114Y/ R122F/K154R/N159D/ Q175K G13Y/K99Y/T104W/ 46,939 79T110A/Y113D/N114D/ R122F/K154R/N159D/ Q175K G13Y/I77L/K99Y/ 48,177 75T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77M/K99Y/ 28,412 46T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77S/K99Y/ 12,003 20T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77V/K99Y/ 35,907 45T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77Y/K99Y/ 33,236 37T104W/ T110A/Y113D/ R122F/ K154R/N159D/ Q175L

Example 4 Baking Performance of Mutants

Baking was done using a scale-down of the Danish Roll recipe (Table 3),using either wheat flour or wheat whole meal flour.

TABLE 3 Recipe used for production of bread. Mini skala Ingredients mlor g Flour 50 Dry yeast 1 Salt 0.8 Sugar 0.8 Water 400 BU-2% Note: Wateris the water absorption @ 400 BU determined by Farinograph analysis offlour (i.e, 400 bakers absorbance-water added according to waterabsorbtion determination using a Brabrender Farinograph, Brabender,Germany). If enzymes are added to the dough, they are added as liquidsolution and by substitution of the same amount of water.Dough Making and Baking

The flour and dry ingredients were mixed for one minute in a 50 gramFarinograph (Brabender, Duisburg, Germany), hereafter water was addedand mixing was continued for another five minutes.

After mixing, four dough lumps were weighed out, each containing10-grams of flour. These were moulded into bread using a hand moulder.Loaves were put into baking pans and placed in a sealed container (witha lid) and left to rest at room temperature for 10 minutes. Hereafter,breads were proofed at 34° C., 85% relative humidity (RH), for 45minutes and finally baked at 230° C. for five minutes in a Bago oven(Bago-line, Fåborg, Denmark).

The breads were cooled for 20 minutes before evaluation (weighing,volume measurement, crumb and crust evaluation).

TABLE 4 Baking performance of mutants-bread volume (ml/g) and relativevolume increase compared to control (no enzyme added) and BS3 (SEQ IDNo. 1 with the modifications D11F and R122D) which show superior bakingperformance compared to the Bacillus sub. XynA wildtype xylanase (SEQ IDNo. 1). Bread vol @ Relative vol. Relative volume Modifications made to0.04 mg/kg Increase increase Seq ID No 1 flour vs control, % vs. BS3, %G13Y/G34K/T110A/ 4.22 41.89 20 Y113D/R122D/Q175L G13Y/K99Y/T104W/ 2.9021.93 7.54 T110A/Y113D/R122F/ K154R/N159D/Q175K G13Y/V81I/K99Y/ 2.8018.02 4.09 T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/2.82 18.86 4.83 T110A/Y113D/R122F/ K154R/N159D/Y166F/ Q175LG13Y/T110A/Y113D/ 2.81 18.08 4.15 R122D/K154R/N159D/ Q175LG13Y/K99Y/T104W/ 2.89 21.74 7.24 T110A/Y113D/R122F/ K154R/N159D/Q175LG13Y/T110A/Y113D/ 2.75 13.55 1.99 R122D/S162E/Q175L G13Y/T110A/Y113D/2.82 15.54 4.48 R122D/S162D/Q175L G13Y/T110A/Y113D/ 2.78 14.02 3.10R122D/W164F/Q175L G13Y/K99Y/T104W/ 2.81 16.44 4.11 T110A/Y113D/N114D/R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 2.73 13.26 1.26T110A/Y113D/N114Y/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 2.80 16.223.74 T110A/Y113D/N114F/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/ 2.8317.58 4.95 T110A/Y113D/I118V/ R122F/K154R/N159D/ Q175L G13Y/K99Y/T104W/2.89 20.65 7.34 T110A/Y113D/N114Y/ R122F/K154R/N159D/ Q175KG13Y/K99Y/T104W/ 2.77 14.73 2.63 T110A/Y113D/N114D/ R122F/K154R/N159D/Q175K G13Y/I77L/K99Y/ 2.81 15.08 4.32 T104W/T110A/Y113D/R122F/K154R/N159D / Q175L G13Y/I77M/K99Y / 2.70 12.43 0.17T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77S/K99Y/ 2.53 5.40(6.09) T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77V/K99Y/ 2.608.19 (3.63) T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L G13Y/I77Y/K99Y/2.73 13.81 1.38 T104W/T110A/Y113D/ R122F/K154R/N159D/ Q175L

Example 5 Effect of Modification to Residue 110 in the Bacillus subtilisXynA Xylanase (SEQ ID NO. 1) or Equivalent Position in Other Family 11Xylanases

Xylanases:

Xylanases mutated in this example are the Bacillus subtilis XynAwildtype xylanase (SEQ ID No. 1), a variant of the Trichoderma reeseiXyn2 xylanase (SEQ ID No. 2) and the Thermomyces lanuginosus XynAwildtype xylanase (SEQ ID No. 3).

The residue mutated is T110 in the Bacillus subtilis XynA wildtypexylanase (SEQ ID No. 1), the equivalent position, T120, in Trichodermareesei xylanase (SEQ ID No. 2) and the equivalent position, T120, inThermomyces lanuginosus XynA wildtype xylanase (SEQ ID No. 3). Thefollowing mutations were made in Bacillus subtilis XynA wildtypexylanase (SEQ ID No. 1): T110H, T110Y, T110S, T110R, T110F, T110Q,T110G, T110K, T110L, T110M, T110I, T110T, T110N, T110E, T110W, T110A andT110C. In both Trichoderma reesei xylanase (SEQ ID No. 2) andThermomyces lanuginosus XynA wildtype xylanase (SEQ ID No. 3) theequivalent position, T120, was mutated. The mutations made is to theamino acid alanine.

Mutations, expression, purification and determination of specificactivity of the wildtype xylanases or their variants are carried out asdescribed in Examples 1, 2 and 3. Except for the purification of theTrichoderma reesei variant xylanase, the Thermomyces lanuginosus XynAwildtype xylanase and their T120A variant, here the purificationprotocol was modified to reflect their pI.

TABLE 5 Specific activity determined as XU/mg xylanase protein of theBacillus subtilis XynA wildtype xylanase (SEQ ID No. 1), the Bacillussubtilis XynA variant xylanase (T110A), the Trichoderma reesei xylanase(SEQ ID No. 2), the Trichoderma reesei Xyn2 variant xylanase (T120A),the Thermomyces lanuginosus XynA wildtype xylanase (SEQ ID NO. 3) andthe Thermomyces lanuginosus XynA variant xylanase (T120A). XylanaseSpecific activity, XU/mg Bacillus subtilis XynA wildtype 23,000 xylanase(SEQ ID No. 1) Bacillus subtilis XynA variant  25874 xylanase (T110A)Bacillus subtilis XynA variant  23104 xylanase (T110N) Bacillus subtilisXynA variant  24782 xylanase (T110E) Bacillus subtilis XynA variant 25478 xylanase (T110W) Bacillus subtilis XynA variant  26390 xylanase(T110C) Trichoderma reesei xylanase 17,500 (SEQ ID No. 2) Trichodermareesei variant  36797 xylanase (T120A) Thermomyces lanuginosus XynA31,300 wildtype xylanase (SEQ ID No 3) Thermomyces lanuginosus XynA 36066 variant xylanase (T120A)

In all cases, the mutation T110A in the Bacillus subtilis XynA wildtypexylanase (SEQ ID No. 1) or the equivalent position (T120) in theTrichoderma reesei xylanase (SEQ ID No. 2) or the Thermomyceslanuginosus XynA wildtype xylanase (SEQ ID No 3) results in asignificant increased specific activity.

In Bacillus subtilis XynA wildtype xylanase (SEQ ID No. 1) also themutations T110E, T110N, T110W, and T110C results in an increasedspecific activity.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

Example 6 Activity of Xylanase Variants on Water-Insoluble SubstrateVersus Insoluble Substrate

Xylanase variants of the BACSU_XynA and TRIRE_Xyn2 was generated usingsite-directed mutagenesis of xylanases and expression in E. coli.

Assay to Determine Activity on Water Unextractable Substrate, WU-AX Act.(Insoluble Substrate):

Samples were diluted in citric acid (0.1 M)—di-sodium-hydrogen phosphate(0.2 M) buffer, pH 5.0, to obtain approx. OD₅₉₀=0.7 in this assay. Threedifferent dilutions of the sample were pre-incubated for 5 minutes at40° C. At time=5 minutes, 1 Xylazyme tablet (crosslinked, dyed xylansubstrate, Megazyme, Bray, Ireland) was added to the enzyme solution ina reaction volume of 1 ml. At time=15 minutes the reaction wasterminated by adding 10 ml of 2% TRIS/NaOH, pH 12. Blanks were preparedusing 1000 μl buffer instead of enzyme solution. The reaction mixturewas centrifuged (1500×g, 10 minutes, 20° C.) and the OD of thesupernatant was measured at 590 nm. One xylanase unit (WU-AX act) isdefined as the xylanase activity increasing OD₅₉₀ with 0.025 per minute.

The substrate (cross-linked and dyed arabinoxylan extracted from wheat)used in the above assay is a good approximate to the correspondingsubstrate in commercial applications.

The following assay was used to determine activity on Water extractablesubstrate, WE-AX act (soluble substrate).

The method used is a modified version of the method described by Lever(Lever, M. Analytical Biochemistry. 47, 273-279, 1972). Soluble wheatarabinoxylan (medium viscosity, obtainable from Megazyme, Bray, Ireland)was used as substrate in a buffer system containing 50 mM NaOAc, pH 5.Substrate concentration was 0.5%. Xylanase activity was measured byquantifying the formation of reducing ends using PAHBAH reagens. Theamount of reducing ends formed and hereby the xylanase activity wasdetermined from a xylose standard curve. Here referred to as WE-AX act.

Backbones Used for Developing New Variants:

Table 6 show xylanase variants backbones used. Y5 corresponds to SEQ IDNO. 2.

ID Variant #154 BACSU_XynA-G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L #160 BACSU_XynA-G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L Y5 TRIRE_Xyn2-T2C/T28C/K58R/+191DY5- TRIRE_Xyn2-T2C/T28C/K58R/T120A/+191D T120AThe Mutations Introduced and the Results Obtained are Illustrated inTable 7

Table 7. Mutations introduced and results obtained. The backbones usedare in bold.

Mutant WU-AX act WE-AX act WU-AX/WE-AX #154/N141Q 1.965 13 146#154/N54Q/N141Q 1.611 10 159 #160/N54Q 1.203 7 161 #160/N141Q 1.785 10175 #154/N54W/N141Q 824 7 118 #160/N54W/N141Q 1.005 6 169 Y5/S63W 918 2536 Y5 35.550 1.487 24 #154 10.350 106 98 #160 5.400 34 157

SEQUENCE LISTING (amino acids in bold are theamino acid, which corresponds to T110 of SEQ ID No. 1):The amino acid sequence of the mature Bacillussubtilis wildtype xylanase (SEQ ID No 1):ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNHVNAWKSHGMNLGSNWAYQVMA TEGYQSSGSSNVTVWThe amino acid sequence of the mature Trichodermareesei xylanase (SEQ ID No 2), also referred to herein as Y5:QCIQPGTGYNNGYFYSYWNDGHGGVTYCNGPGGQFSVNWSNSGNFVGGKGWQPGTKNRVINFSGSYNPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVSDThe amino acid sequence of the mature Thermomyceslanuginosus XynA wildtype xylanase (SEQ ID No. 3):QTTPNSEGWHDGYYYSWWSDGGAQATYTNLEGGTYEISWGDGGNLVGGKGWNPGLNARAIHFEGVYQPNGNSYLAVYGWTRNPLVEYYIVENFGTYDPSSGATDLGTVECDGSIYRLGKTTRVNAPSIDGTQTFDQYWSVRQDKRTSGTVQTGCHFDAWARAGLNVNGDHYYQIVATEGYFSSGYARITVADVGThe amino acid sequence of the mature Streptomycesviridosporus xylanase (Seq ID No 4):WTDAQGTVSMDLGSGGTYSTQWRNTGNFVAGKGWSTGGRKTVNYSGTFNPSGNAYLTLYGWTTGPLIEYYIVDNWGTYRPTGKYKGTVTSDGGTYDIYKTTRYNAPSIEGTKTFDQYWSVRQSKRTGGTITSGNHFDAWARNGMNLGNHN YMIMATEGYQSSGSSTITVSeq ID No 5 (gi|139868|sp|P18429.1|XYNA BACSU RecName: Full =Endo-1,4-beta-xylanase A; Short = Xylanase A; AltName: Full =1,4-beta-D-xylan xylanohydrolase A):MFKFKKNFLVGLSAALMSISLFSATASAASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNHVNAWKSHGMNLGSNWAYQVMATE GYQSSGSSNVTVWSeq ID No 6 (gi|2302074|emb|CAA03092.1| unnamedprotein product [unidentified]):MRQKKLTLILAFLVCFALTLPAEIIQAQIVTDNSIGNHDGYDYEFWKDSGGSGTMILNHGGTFSAQWNNVNNILFRKGKKFNETQTHQQVGNMSINYGANFQPNGNAYLCVYGWTVDPLVEYYIVDSWGNWRPPGATPKGTITVDGGTYDIYETLRVNQPSIKGIATFKQYWSVRRSKRTSGTISVSNHFRAWENLGMNMGKMYEVALTVEGYQSSGSANVYSNTLRINGNPLSTISNDESITLDKNNSeq ID No 7 (gi|167246404|gb|ABZ24364.1| Sequence5 from U.S. Pat. No. 7,314,743):MVSFTSLLAASPPSRASCRPAAEVESVAVEKRQTIQPGTGYNNGYFYSYWNDGHGGVTYTNGPGGQFSVNWSNSGNFVGGKGWQPGTKNKVINFSGSYNPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVS Seq ID No 8 (gi|5969551|gb|AAE10889.1| Sequence 2from U.S. Pat. No. 5,817,500):MVGFTPVALAALAATGALAFPAGNATELEKRQTTPNSEGWHDGYYYSWWSDGGAQATYTNLEGGTYEISWGDGGNLVGGKGWNPGLNARAIHFEGVYQPNGNSYLAVYGWTRNPLVEYYIVENFGTYDPSSGATDLGTVECDGSIYRLGKTTRVNAPSIDGTQTFDQYWSVRQDKRTSGTVQTGCHFDAWARAGLNVNGDHYYQIVATEGYFSSGYARITVADVG Seq ID No 9 (gi|76059070|emb|CAJ30753.1|unnamed protein product [Paenibacillus pabuli]):MFKFGKKLLTVVLAASMSFGVFAATTGATDYWQNWTDGGGTVNAVNGSGGNYSVNWQNTGNFVVGKGWTYGTPNRVVNYNAGVFSPSGNGYLTFYGWTRNALIEYYVVDNWGTYRPTGTYKGTVTSDGGTYDIYTTMRYNQPSIDGYSTFPQYWSVRQSKRPIGVNSQITFQNHVNAWASKGMYLGNSWSYQVMATEGYQ SSGSSNVTVWSeq ID No 10 (gi|74197761|emb|CAJ29666.1| unnamedprotein product [Bacillus halodurans]):MFKFVTKVLTVVIAATISFCLSAVPASANTYWQYWTDGGGTVNATNGPGGNYSVTWRDTGNFVVGKGWEIGSPNRTIHYNAGVWEPSGNGYLTLYGWTRNQLIEYYVVDNWGTYRPTGTHRGTVVSDGGTYDIYTTMRYNAPSIDGTQTFQQFWSVRQSKRPTGNNVSITFSNHVNAWRNAGMNLGSSWSYQVLATEGYQ SSGRSNVTVWSeq ID No 11 (gi|4756811|emb|CAB42305.1| unnamedprotein product [unidentified]):MRQKKLTFILAFLVCFALTLPAEIIQAQIVTDNSIGNHDGYDYEFWKDSGGSGTMILNHGGTFSAQWNNVNNILFRKGKKFNETQTHQQVGNMSINYGANEFQPNGNAYLCVYGWTVDPLVYYIVDSWGNWRPPGATPKGTITVDGGTYDIYETLRVNQPSIKGIATFKQYWSVRRSKRTSGTISVSNHFRAWENLGMNMGKMYEVALTVEGYQSSGSANVYSNTLRINGNPLSTISNDKSITLDKNNSeq ID No 12 (gi|2293951|emb|CAA02246.1| unnamedprotein product [Bacillus subtilis] Bacillussubtilis: (U.S. Pat. No. 5,306,633)):MFKFKKKFLVGLTAAFMSISMFSATASAAGTDYWQNWTDGGGTVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDNTTFTQYWSVRQSKRPTGSNAAITFSNHVNAWKSHGMNLGSNWAYQVLATE GYKSSGSSNVTVWSeq ID No 13 (gi|42688917|gb|AAS31735.1| Sequence14 from U.S Pat. No. 6,682,923):MNLRKLRLLFVMCIGLTLILTAVPAHARTITNNEMGNHSGYDYELWKDYGNTSMTLNNGGAFSAGWNNIGNALFRKGKKFDSTRTHHQLGNISINYNASFNPGGNSYLCVYGWTQSPLAEYYIVDSWGTYRPTGAYKGSFYADGGTYDIYETTRVNQPSIIGIATFKQYWSVRQTKRTSGTVSVSAHFRKWESLGMPMGKMYETAFTVEGYQSSGSANVMTNQLFIGN Seq ID No 14 (gi|10040204|emb|CAC07798.1|unnamed protein product [Penicillium funiculosum]):MKLFLAAIVLCATAATAFPSELAQRAAGDLSKRQSITTSQTGTNNGYYYSFWTNGGGEVTYTNGDNGEYSVTWVDCGDFTSGKGWNPANAQTVTYSGEFNPSGNAYLAVYGWTTDPLVEYYILESYGTYNPSSGLTSLGQVTSDGGTYDIYSTQRVNQPSIEGTSTFNQYWSVRTEKRVGGTVTTANHFAAWKALGLEMGTYNYMIVSTEGYESSGSSTITVS Seq ID No 15 (gi|2302074|emb|CAA03092.1| unnamedprotein product [unidentified]):QIVTDNSIGNHDGYDYEFWKDSGGSGTMILNHGGTFSAQWNNVNNILFRKGKKFNETQTHQQVGNMSINYGANFQPNGNAYLCVYGWTVDPLVEYYIVDSWGNWRPPGATPKGTITVDGGTYDIYETLRVNQPSIKGIATFKQYWSVRRSKRTSGTISVSNHFRAWENLGMNMGKMYEVALTVEGYQSSGSANVYSNTLR INGNPLSTISNDESITLDKNNSeq ID No 16 (gi|167246404|gb|ABZ24364.1| Sequence5 from U.S Pat. No. 7,314,743):QTIQPGTGYNNGYFYSYWNDGHGGVTYTNGPGGQFSVNWSNSGNFVGGKGWQPGTKNKVINFSGSYNPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVSSeq ID No 17 (gi|76059070|emb|CAJ30753.1| unnamedprotein product [Paenibacillus pabuli]):TDYWQNWTDGGGTVNAVNGSGGNYSVNWQNTGNFVVGKGWTYGTPNRVVNYNAGVFSPSGNGYLTFYGWTRNALIEYYVVDNWGTYRPTGTYKGTVTSDGGTYDIYTTMRYNQPSIDGYSTFPQYWSVRQSKRPIGVNSQITFQNHVNAWASKGMYLGNSWSYQVMATEGYQSSGSSNVTVWSeq ID No 18 (gi|74197761|emb|CAJ29666.1| unnamedprotein product [Bacillus halodurans]):NTYWQYWTDGGGTVNATNGPGGNYSVTWRDTGNFVVGKGWEIGSPNRTIHYNAGVWEPSGNGYLTLYGWTRNQLIEYYVVDNWGTYRPTGTHRGTVVSDGGTYDIYTTMRYNAPSIDGTQTFQQFWSVRQSKRPTGNNVSITFSNHVNAWRNAGMNLGSSWSYQVLATEGYQSSGRSNVTVWSeq ID No 19 (gi|4756811|emb|CAB42305.1| unnamed protein product):QIVTDNSIGNHDGYDYEFWKDSGGSGTMILNHGGTFSAQWNNVNNILFRKGKKFNETQTHQQVGNMSINYGANFQPNGNAYLCVYGWTVDPLVEYYIVDSWGNWRPPGATPKGTITVDGGTYDIYETLRVNQPSIKGIATFKQYWSVRRSKRTSGTISVSNHFRAWENLGMNMGKMYEVALTVEGYQSSGSANVYSNTLR INGNPLSTISNDKSITLDKNNSeq ID No 20 (gi|2293951|emb|CAA02246.1| unnamedprotein product [Bacillus subtilis] Bacillussubtilis: (U.S. Pat. No. 5,306,633)):AGTDYWQNWTDGGGTVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDNTTFTQYWSVRQSKRPTGSNAAITFSNHVNAWKSHGMNLGSNWAYQVLATEGYKSSGSSNVTVWSeq ID No 21 (gi|42688917|gb|AAS31735.1| Sequence14 from U.S. Pat. No. 6,682,923):RTITNNEMGNHSGYDYELWKDYGNTSMTLNNGGAFSAGWNNIGNALFRKGKKFDSTRTHHQLGNISINYNASFNPGGNSYLCVYGWTQSPLAEYYIVDSWGTYRPTGAYKGSFYADGGTYDIYETTRVNQPSIIGIATFKQYWSVRQTKRTSGTVSVSAHFRKWESLGMPMGKMYETAFTVEGYQSSGSANVMTNQLFIG NSeq ID No 22 (gi|10040204|emb|CAC07798.1| unnamedprotein product [Penicillium funiculosum]):AFPSELAQRAAGDLSKRQSITTSQTGTNNGYYYSFWTNGGGEVTYTNGDNGEYSVTWVDCGDFTSGKGWNPANAQTVTYSGEFNPSGNAYLAVYGWTTDPLVEYYILESYGTYNPSSGLTSLGQVTSDGGTYDIYSTQRVNQPSIEGTSTFNQYWSVRTEKRVGGTVTTANHFAAWKALGLEMGTYNYMIVSTEGYESSG SSTITVSSEQ ID No 23 shows the amino acid sequence of themature Bacillus subtilis xylanase variant, BS3(wildtype with D11F/R122D mutations):ASTDYWQNWTFGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDDTTFTQYWSVRQSKRPTGSNATITFSNHVNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVWSeq ID No 24 shows the sequence of the maturewheat xylanase inhibitor sequence:MPPVLLLVLAASLVALPSCQSLPVLAPVTKDPATSLYTIPFHDGASLVLDVAGPLVWSTCDGGQPPAEIPCSSPTCLLANAYPAPGCPAPSCGSDKHDKPCTAYPYNPVSGACAAGSLSHTRFVANTTDGSKPVSKVNVGVLAACAPSKLLASLPRGSTGVAGLANSGLALPAQVASAQKVANRFLLCLPTGGPGVAIFGGGPVPWPQFTQSMPYTPLVTKGGSPAHYISARSIVVGDTRVPVPEGALATGGVMLSTRLPYVLLRPDVYRPLMDAFTKALAAQHANGAPVARAVEAVAPFGVCYDTKTLGNNLGGYAVPNVQLGLDGGSDWTMTGKNSMVDVKQGTACVAFVEMKGVAAGDGRAPAVILGGAQMEDFVLDFDMEKKRLGFSRLPHFTGCG GLSeq ID No 25 (sequence 11 of U.S. Pat. No. 6,682,923):ASTDWWENWTIGGGIVNAVNGSGGNYSVNWSNTGNFDVAKGWTTGSPFRTINYNAGVWAPNGWGELELYGWTRSPLIEYLVVDSWGTNRPTGTYKGTVKSDGGTYDIYTDTRYNYPSEDGDRTTMTQYSSVRQSKRPTGSNATITFTNHVNAWKSHGMNLGSNWAYQDMATEGYQSSGSSNVTVW

Embodiments of the Invention

1. A polypeptide having xylanase activity and comprising an amino acidsequence, said amino acid sequence having at least 88% identity with SEQID No. 1 or having at least 75% identity with an amino acid sequenceselected from SEQ ID No. 2-22, and which polypeptide has an amino acidmodification in position 110, wherein said position 110 is determined asthe position corresponding to position 110 of B. subtilis xylanasesequence shown as SEQ ID No. 1 by alignment.

2. A polypeptide having xylanase activity and comprising an amino acidsequence, said amino acid sequence having at least 88% identity with SEQID No. 1 and which polypeptide has an amino acid modification inposition 110, wherein said position 110 is determined as the positioncorresponding to position 110 of B. subtilis xylanase sequence shown asSEQ ID No. 1 by alignment.

3. A polypeptide having xylanase activity and comprising an amino acidsequence, said amino acid sequence having at least 75% identity with SEQID No. 2 and which polypeptide has an amino acid modification inposition 110, wherein said position 110 is determined as the positioncorresponding to position 110 of B. subtilis xylanase sequence shown asSEQ ID No. 1 by alignment.

4. A polypeptide having xylanase activity and comprising an amino acidsequence, said amino acid sequence having at least 75% identity with SEQID No. 3 and which polypeptide has an amino acid modification inposition 110, wherein said position 110 is determined as the positioncorresponding to position 110 of B. subtilis xylanase sequence shown asSEQ ID No. 1 by alignment.

5. The polypeptide according to embodiment 2, wherein said polypeptidehas at least 90, 92 or 95% identity with SEQ ID No. 1.

6. The polypeptide according to any one of the embodiments 1 and 3-4,wherein said polypeptide has at least 76, 78, 80, 85, 90, 95, 98 or 95%identity with the sequence with which is has the highest percentage ofidentity selected from SEQ ID No. 2-22.

7. The polypeptide according embodiment 3, wherein said polypeptide hasat least 76, 78, 80, 85, 90, 95, 98 or 95% identity with SEQ ID No. 2.

8. The polypeptide according embodiment 4, wherein said polypeptide hasat least 76, 78, 80, 85, 90, 95, 98 or 95% identity with SEQ ID No. 3.

9. The polypeptide according to any one of the embodiments 1-6 having a3-jelly roll fold.

10. The polypeptide according to any one of the embodiments 1-9, whereinthe amino acid modification in position 110 is an amino acidsubstitution.

11. The polypeptide according to any one of the embodiments 1-10,wherein the amino acid modification in position 110 is a substitution toany one different amino acid residue selected from the group consistingof: alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, tryptophan, tyrosine andvaline.

12. The polypeptide according to any one of the embodiments 1-11,wherein the amino acid modification in position 110 is a substitution toany one different amino acid residue selected from the group consistingof: glutamic acid, tryptophan, alanine and cysteine.

13. The polypeptide according to embodiment 12, wherein the amino acidmodification in position 110 is a substitution to alanine.

14. The polypeptide according to any one of the embodiments 1-13 havinga total number of amino acids of less than 250, such as less than 240,such as less than 230, such as less than 220, such as less than 210,such as less than 200 amino acids, such as in the range of 160 to 240,such as in the range of 160 to 220 amino acids.

15. The polypeptide according to any one of the embodiments 1-14,comprising one or more modification(s) at any one or more of amino acidpositions: 11, 12, 13, 34, 54, 77, 81, 99, 104, 113, 114, 118, 122, 141,154, 159, 162, 164, 166 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

16. The polypeptide according to any one of the embodiments 1-15,comprising one or more amino acid substitutions selected from the groupconsisting of: 11F, 12F, 54Q, 54W, 122D, 113A, 13Y, 113D, 141Q, 175L,122F, 34K, 99Y, 104W, 154R, 159D, 175K, 81I, 166F, 162E, 162D, 164F,114D, 114Y, 114F, 118V, 175K, 77L, 77M, 77S, 77V, and 77Y, theposition(s) being determined as the corresponding position of B.subtilis amino acid sequence shown as SEQ ID No. 1.

17. The polypeptide according to any one of the embodiments 2, 5, 9-16,comprising one or more amino acid substitutions selected from the groupconsisting of: D11F, G12F, N54Q, R122D, Y113A, G13Y, Y113D, N141Q,Q175L, R122F, G34K, K99Y, T104W, K154R, N159D, Q175K, V81I, Y166F,S162E, S162D, W164F, N114D, N114Y, N114F, I118V, I77L, I77M, I77S, I77V,and I77Y, the position(s) being determined as the corresponding positionof B. subtilis amino acid sequence shown as SEQ ID No. 1.

18. The polypeptide according to any one of the embodiments 1-17,comprising one or more modification(s) at any one or more of amino acidpositions: 13, 99, 104, 113, 122, 154, 159 and 175, the position(s)being determined as the corresponding position of B. subtilis amino acidsequence shown as SEQ ID No. 1.

19. The polypeptide according to any one of the embodiments 1-18,comprising substitution(s) at the amino acid positions: 13, 99, 104,113, 122, 154, 159 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

20. The polypeptide according to any one of the embodiments 18-19,further comprising one or more modification(s) at any one or more ofamino acid positions: 114 and 166, the position(s) being determined asthe corresponding position of B. subtilis amino acid sequence shown asSEQ ID No. 1.

21. The polypeptide according to any one of the embodiments 18-19,further comprising one or more substitution(s) at any one or more ofamino acid positions: 114 and 166, the position(s) being determined asthe corresponding position of B. subtilis amino acid sequence shown asSEQ ID No. 1.

22. The polypeptide according to any one of the embodiments 18-19,comprising substitution(s) in at least at four of the following aminoacid positions: 13, 99, 104, 113, 114, 122, 154, 159, 166, and 175, theposition(s) being determined as the corresponding position of B.subtilis amino acid sequence shown as SEQ ID No. 1.

23. The polypeptide according to any one of the embodiments 18-22,comprising substitution(s) at the amino acid positions: 13, 99, 104,113, 114, 122, 154, 159 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

24. The polypeptide according to any one of the embodiments 18-22,comprising substitution(s) at the amino acid positions: 13, 99, 104,113, 122, 154, 159, 166 and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

25 The polypeptide according to any one of the embodiments 18-20,comprising substitution(s) at the amino acid positions: 13, 99, 104,113, 122, 154, 159, and 175, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

26. The polypeptide according to any one of the embodiments 22-25,comprising one or more amino acid substitutions selected from the groupconsisting of: 13Y, 99Y, 104W, 110A, 113D, 114D, 114F, 122F, 154R, 159D,166F, 175K, and 175L, the position(s) being determined as thecorresponding position of B. subtilis amino acid sequence shown as SEQID No. 1.

27. The polypeptide according to any one of the embodiments 1-26,wherein the amino acid sequence of said polypeptide has at least five,six, seven, eight, nine or ten amino acid substitutions compared to thesequence selected among SEQ ID No. 1-22 with which it has the highestidentity.

28. The polypeptide according to embodiment 27, wherein the amino acidsequence of said polypeptide has at least nine or ten amino acidsubstitutions.

29. The polypeptide according to any one of the embodiments 1-28,comprising one or more amino acid substitutions selected from the groupconsisting of:

-   a. D11F, R122D and T110A;-   b. D11F, R122D, T110A and Y113A;-   c. G13Y, T110A, Y113D, R122D and Q175L;-   d. G13Y, T110A, Y113D, R122F and Q175L;-   e. G13Y, G34K, T110A, Y113D, R122D and Q175L;-   f. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and Q175K;-   g. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D, Q175 and    V81I;-   h. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D, Y166F and    Q175L;-   i. G13Y, T110A, Y113D, R122D, K154R, N159D and Q175L;-   j. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and Q175L;-   k. G13Y, T110A, Y113D, R122D, S162E and Q175L;-   l. G13Y, T110A, Y113D, R122D, S162D and Q175L;-   m. G13Y, T110A, Y113D, R122D, W164F and Q175L;-   n. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175L;-   o. G13Y, K99Y, T104W, T110A, Y113D, N114Y, R122F, K154R, N159D and    Q175L;-   p. G13Y, K99Y, T104W, T110A, Y113D, N114F, R122F, K154R, N159D and    Q175L;-   q. G13Y, K99Y, T104W, T110A, Y113D, I118V, R122F, K154R, N159D and    Q175L;-   r. G13Y, K99Y, T104W, T110A, Y113D, N114Y, R122F, K154R, N159D and    Q175K;-   s. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175K;-   t. G13Y, I77L, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   u. G13Y, I77M, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    175L;-   v. G13Y, I77S, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   w. G13Y, I77V, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L;-   x. G13Y, I77Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and    Q175L-   y. G13Y, K99Y, T104W, T110A, Y113D, R122F, N141Q, K154R, N159D, and    Q175L;-   z. G13Y, N54Q, K99Y, T104W, T110A, Y113D, R122F, N141Q, K154R,    N159D, and Q175;-   aa. G13Y, N54W, K99Y, T104W, T110A, Y113D, R122F, 141Q, K154R,    N159D, and 175L;-   bb. G13Y, N54Q, K99Y, T104W, T110A, Y113D, N114F, R122F, K154R,    N159D, and Q175L;-   cc. G13Y, K99Y, T104W, T110A, Y113D, N114F, R122F, 141Q, K154R,    N159D, and Q175L;-   dd. G13Y, 54Q, K99Y, T104W, T110A, Y113D, N114F, R122F, 141Q, K154R,    N159D, and Q175L;    the position(s) being determined as the corresponding position of B.    subtilis amino acid sequence shown as SEQ ID No. 1.

30. The polypeptide according to any one of the embodiments 1-29,comprising one or more amino acid substitutions selected from the groupconsisting of:

-   a. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175K;-   b. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D, Y166F and    Q175L;-   c. G13Y, K99Y, T104W, T110A, Y113D, R122F, K154R, N159D and Q175L;-   d. G13Y, K99Y, T104W, T110A, Y113D, N114D, R122F, K154R, N159D and    Q175L;-   e. G13Y, K99Y, T104W, T110A, Y113D, N114F, R122F, K154R, N159D and    Q175L;    the position(s) being determined as the corresponding position of B.    subtilis amino acid sequence shown as SEQ ID No. 1.

31. The polypeptide according to any one of the embodiments 1-30 havingbran solubilisation activity.

32. The polypeptide according to any one of the embodiments 1-31 inisolated form.

33. The polypeptide according to any one of the embodiments 1-32,wherein the amino acid modification in position 110 is not T110D.

34. The polypeptide according to any one of embodiments 1-33 having animproved xylanase activity compared to the B. subtilis amino acidsequence shown as SEQ ID No. 1 as measured in a xylanase activity assay.

35. The polypeptide according to any one of embodiments 1-34 having animproved xylanase activity as a result of the modification in position110.

36. The polypeptide according to any one of embodiments 1-35 having animproved bran solubilisation activity compared to the B. subtilis aminoacid sequence shown as SEQ ID No. 1 as measured in a bran solubilisationactivity assay.

37. The polypeptide according to any one of embodiments 1-36 having animproved bran solubilisation activity as a result of the modification inposition 110.

38. The polypeptide according to any one of embodiments 1-37 having areduced sensitivity to a xylanase inhibitor.

39. The polypeptide according to any one of embodiments 1-38, whereinsaid polypeptide has an amino acid sequence comprising modifications atpositions selected from the list consisting of:

-   -   a) 13/110/113/122/154/159/175;    -   b) 13/99/104/110/113/122/154/159/166/175;    -   c) 13/99/104/110/113/114/122/154/159/175;    -   d) 13/110/113/122/175;    -   e) 13/99/104/110/113/122/154/159/175;    -   f) 13/99/104/110/113/122/154/159/175;    -   g) 13/99/104/110/113/114/122/154/159/175;    -   h) 13/99/104/110/113/114/122/154/159/175;    -   i) 13/99/104/110/113/114/122/154/159/175;    -   j) 13/99/104/110/113/114/122/154/159/175;    -   k) 13/77/99/104/110/113/122/154/159/175;    -   l) 13/81/99/104/110/113/122/154/159/175;    -   m) 13/110/113/122/164/175;    -   n) 13/110/113/122/162/175;    -   o) 13/110/113/122/175;    -   p) 11/122/110/113;    -   q) 13/77/99/104/110/113/122/154/159/175;    -   r) 11/122/110;    -   s) 13/34/110/113/122/175;    -   t) 13/77/99/104/110/113/122/154/159/175;    -   u) 13/77/99/104/110/113/122/154/159/175;    -   v) 13/99/104/110/113/118/122/154/159/175;    -   w) 13/110/113/122/162/175;    -   x) 13/77/99/104/110/113/122/154/159/175;    -   y) 13/99/104/110/113/122/141/154/159/175;    -   z) 13/54/99/104/110/113/122/141/154/159/175;    -   aa) 13/54/99/104/110/113/122/141/154/159/175;    -   bb) 13/54/99/104/110/113/114/122/154/159/175;    -   cc) 13/99/104/110/113/114/122/141/154/159/175; and    -   dd) 13/54/99/104/110/113/114/122/141/154/159/175,        the position(s) being determined as the corresponding position        of subtilis amino acid sequence shown as SEQ ID No. 1.

40. The polypeptide according to any one of embodiments 1-39, whereinsaid polypeptide has an amino acid sequence comprising amino acidsubstitutions selected from the list consisting of:

-   -   a) 13Y/110A/113D/122D/154R/159D/175L;    -   b) 13Y/99Y/104W/110A/113D/122F/154R/159D/166F/175L;    -   c) 13Y/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   d) 13Y/110A/113D/122F/175L;    -   e) 13Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   f) 13Y/99Y/104W/110A/113D/122F/154R/159D/175K;    -   g) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175K;    -   h) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175L;    -   i) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175L;    -   j) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175K;    -   k) 13Y/77L/99Y/104W/110A/113D/122F/154R/159D/175L;    -   l) 13Y/81I/99Y/104W/110A/113D/122F/154R/159D/175L;    -   m) 13Y/110A/113D/122D/164F/175L;    -   n) 13Y/110A/113D/122D/162D/175L;    -   o) 13Y/110A/113D/122D/175L;    -   p) 11F/122D/110A/113A;    -   q) 13Y/77Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   r) 11F/122D/110A;    -   s) 13Y/34K/110A/113D/122D/175L;    -   t) 13Y/77V/99Y/104W/110A/113D/122F/154R/159D/175L;    -   u) 13Y/77M/99Y/104W/110A/113D/122F/154R/159D/175L;    -   v) 13Y/99Y/104W/110A/113D/118V/122F/154R/159D/175L;    -   w) 13Y/110A/113D/122D/162E/175L;    -   x) 13Y/77S/99Y/104W/110A/113D/122F/154R/159D/175L;    -   y) 13Y/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   z) 13Y/54Q/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   aa) 13Y/54W/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   bb) 13Y/54Q/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   cc) 13Y/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L; and    -   dd) 13Y/54Q/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L;        the position(s) being determined as the corresponding position        of subtilis amino acid sequence shown as SEQ ID No. 1.

41. The polypeptide according to any one of embodiments 1-40, whereinsaid polypeptide has an amino acid sequence of SEQ ID No. 1 comprisingamino acid substitutions selected from the list consisting of

-   -   a) G13Y/T110A/Y113D/R122D/K154R/N159D/Q175L;    -   b) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Y166F/Q175L;    -   c) G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   d) G13Y/T110A/Y113D/R122F/Q175L;    -   e) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   f) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175K;    -   g) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175K;    -   h) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175L;    -   i) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175L;    -   j) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175K;    -   k) G13Y/I77L/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   l) G13Y/V81I/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   m) G13Y/T110A/Y113D/R122D/W164F/Q175L;    -   n) G13Y/T110A/Y113D/R122D/S162D/Q175L;    -   o) G13Y/T110A/Y113D/R122D/Q175L;    -   p) D11F/R122D/T110A/Y113A;    -   q) G13Y/I77Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   r) D11F/R122D/T110A;    -   s) G13Y/G34K/T110A/Y113D/R122D/Q175L;    -   t) G13Y/I77V/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   u) G13Y/I77M/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   v) G13Y/K99Y/T104W/T110A/Y113D/1118V/R122F/K154R/N159D/Q175L;    -   w) G13Y/T110A/Y113D/R122D/S162E/Q175L; and    -   x) G13Y/I77S/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L    -   y) G13Y/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   z)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   aa)        G13Y/N54W/K99Y/T104W/T110A/Y113D/R122F/141Q/K154R/N159D/175L;    -   bb)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   cc)        G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L;        and    -   dd)        G13Y/54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L.

42. The polypeptide according to any one of embodiments 1-41, whereinsaid polypeptide has an amino acid sequence, which consists of aminoacid substitutions selected from the list consisting of:

-   -   a) 13Y/110A/113D/122D/154R/159D/175L;    -   b) 13Y/99Y/104W/110A/113D/122F/154R/159D/166F/175L;    -   c) 13Y/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   d) 13Y/110A/113D/122F/175L;    -   e) 13Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   f) 13Y/99Y/104W/110A/113D/122F/154R/159D/175K;    -   g) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175K;    -   h) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175L;    -   i) 13Y/99Y/104W/110A/113D/114D/122F/154R/159D/175L;    -   j) 13Y/99Y/104W/110A/113D/114Y/122F/154R/159D/175K;    -   k) 13Y/77L/99Y/104W/110A/113D/122F/154R/159D/175L;    -   l) 13Y/81I/99Y/104W/110A/113D/122F/154R/159D/175L;    -   m) 13Y/110A/113D/122D/164F/175L;    -   n) 13Y/110A/113D/122D/162D/175L;    -   o) 13Y/110A/113D/122D/175L;    -   p) 11F/122D/110A/113A;    -   q) 13Y/77Y/99Y/104W/110A/113D/122F/154R/159D/175L;    -   r) 11F/122D/110A;    -   s) 13Y/34K/110A/113D/122D/175L;    -   t) 13Y/77V/99Y/104W/110A/113D/122F/154R/159D/175L;    -   u) 13Y/77M/99Y/104W/110A/113D/122F/154R/159D/175L;    -   v) 13Y/99Y/104W/110A/113D/118V/122F/154R/159D/175L;    -   w) 13Y/110A/113D/122D/162E/175L; and    -   x) 13Y/77S/99Y/104W/110A/113D/122F/154R/159D/175L    -   y) 13Y/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   z) 13Y/54Q/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   aa) 13Y/54W/99Y/104W/110A/113D/122F/141Q/154R/159D/175L;    -   bb) 13Y/54Q/99Y/104W/110A/113D/114F/122F/154R/159D/175L;    -   cc) 13Y/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L; and    -   dd) 13Y/54Q/99Y/104W/110A/113D/114F/122F/141Q/154R/159D/175L,        the position(s) being determined as the corresponding position        of subtilis amino acid sequence shown as SEQ ID No. 1.

43. The polypeptide according to any one of embodiments 1-42, whereinsaid polypeptide has an amino acid sequence of SEQ ID No. 1, whichconsists of amino acid substitutions selected from the list consistingof

-   -   a) G13Y/T110A/Y113D/R122D/K154R/N159D/Q175L;    -   b) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Y166F/Q175L;    -   c) G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   d) G13Y/T110A/Y113D/R122F/Q175L;    -   e) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   f) G13Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175K;    -   g) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175K;    -   h) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175L;    -   i) G13Y/K99Y/T104W/T110A/Y113D/N114D/R122F/K154R/N159D/Q175L;    -   j) G13Y/K99Y/T104W/T110A/Y113D/N114Y/R122F/K154R/N159D/Q175K;    -   k) G13Y/I77L/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   l) G13Y/V81I/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   m) G13Y/T110A/Y113D/R122D/W164F/Q175L;    -   n) G13Y/T110A/Y113D/R122D/S162D/Q175L;    -   o) G13Y/T110A/Y113D/R122D/Q175L;    -   p) D11F/R122D/T110A/Y113A;    -   q) G13Y/I77Y/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   r) D11F/R122D/T110A;    -   s) G13Y/G34K/T110A/Y113D/R122D/Q175L;    -   t) G13Y/I77V/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   u) G13Y/I77M/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L;    -   v) G13Y/K99Y/T104W/T110A/Y113D/1118V/R122F/K154R/N159D/Q175L;    -   w) G13Y/T110A/Y113D/R122D/S162E/Q175L; and    -   x) G13Y/I77S/K99Y/T104W/T110A/Y113D/R122F/K154R/N159D/Q175L    -   y) G13Y/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   z)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/R122F/N141Q/K154R/N159D/Q175L;    -   aa)        G13Y/N54W/K99Y/T104W/T110A/Y113D/R122F/141Q/K154R/N159D/175L,    -   bb)        G13Y/N54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/K154R/N159D/Q175L;    -   cc)        G13Y/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L,        and    -   dd)        G13Y/54Q/K99Y/T104W/T110A/Y113D/N114F/R122F/141Q/K154R/N159D/Q175L.

44. The polypeptide according to any one of embodiments 1-43, which isnot SEQ ID No. 25.

45. A method of identifying a polypeptide according to any one of theembodiments 1-44, said method comprising:

(i) preparing a polypeptide having at least 88% identity with SEQ ID No.1 or having at least 75% identity with an amino acid sequence selectedfrom SEQ ID No. 2-22, and which polypeptide has an amino acidmodification in position 110, wherein said position 110 is determined asthe position corresponding to position 110 of B. subtilis xylanasesequence shown as SEQ ID No. 1 by alignment;(ii) comparing the bran solubilisation and/or xylanase activity of saidpolypeptide with the bran solubilisation and/or xylanase activity of theamino acid sequence selected among SEQ ID NOs: 1-22 with which is hasthe highest percentage of identity; and(iii) selecting the polypeptide if it has improved bran solubilisationand/or improved xylanase activity compared to the amino acid sequenceselected among SEQ ID NOs: 1-22 with which is has the highest percentageof identity.

46. A method of preparing a polypeptide according to any one ofembodiments 1-44, said method comprising expressing a nucleotidesequence encoding said polypeptide; and optionally isolating and/orpurifying the polypeptide after expression.

47. The method according to embodiment 46, wherein said polypeptide isprepared by modifying either a polypeptide amino acid sequence atposition 110 or a codon that encodes an amino acid residue at position110 in a nucleotide sequence encoding a polypeptide amino acid sequence,wherein position 110 is determined with reference to the B. subtilisxylanase sequence shown as SEQ ID No. 1.

48. A nucleotide sequence encoding a polypeptide according to any one ofembodiments 1 to 44.

49. A vector comprising the nucleotide sequence according to embodiment48.

50. A cell that has been transformed with the nucleotide sequence ofembodiment 48 or the vector of embodiment 49.

51. A host organism that has been transformed with the nucleotidesequence of embodiment 48 or the vector of embodiment 49.

52. A composition comprising the polypeptide according to any one ofembodiments 1-44 or a polypeptide identified according to any embodiment45 or a polypeptide prepared according to embodiments 46-47 or thenucleotide sequence according to embodiment 48 or the vector accordingto embodiment 49 or the cell according to embodiment 50 or the organismaccording to embodiment 51 admixed with a non toxic component.

53. A dough comprising the polypeptide according to any one ofembodiments 1-44 or a polypeptide identified according to embodiment 45or a polypeptide prepared according to embodiments 46-47 or thenucleotide sequence according to embodiment 48 or the vector accordingto embodiment 49 or the cell according to embodiment 50 or the organismaccording to embodiment 51 admixed with a non toxic component or acomposition according to embodiment 52.

54. A bakery product comprising the polypeptide according to any one ofembodiments 1-44 or a polypeptide identified according to embodiment 45or a polypeptide prepared according to embodiments 46-47 or thenucleotide sequence according to embodiment 48 or the vector accordingto embodiment 49 or the cell according to embodiment 50 or the organismaccording to embodiment 51 admixed with a non toxic component or acomposition according to embodiment 52 or a dough according toembodiment 53.

55. Animal feed comprising the polypeptide according to any one ofembodiments 1-44 or a polypeptide identified according to embodiment 45or a polypeptide prepared according to embodiments 46-47 or thenucleotide sequence according to embodiment 48 or the vector accordingto embodiment 49 or the cell according to embodiment 50 or the organismaccording to embodiment 51 admixed with a non toxic component or acomposition according to embodiment 52.

56. A cleaning compositions comprising the polypeptide according to anyone of embodiments 1-44 or a polypeptide identified according toembodiment 45 or a polypeptide prepared according to embodiments 46-47.

57. A method of degrading or modifying a plant cell wall which methodcomprises contacting said plant cell wall with the polypeptide accordingto any one of embodiments 1-44 or a polypeptide identified according toembodiment 45 or a polypeptide prepared according to embodiments 46-47or the nucleotide sequence according to embodiment 48 or the vectoraccording to embodiment 49 or the cell according to embodiment 50 or theorganism according to embodiment 51 admixed with a non toxic componentor a composition according to embodiment 52.

58. A method of processing a plant material which method comprisescontacting said plant material with the polypeptide according to any oneof embodiments 1-44 or a polypeptide identified according to embodiment45 or a polypeptide prepared according to embodiments 46-47 or thenucleotide sequence according to embodiment 47 or the vector accordingto embodiment 49 or the cell according to embodiment 50 or the organismaccording to embodiment 51 admixed with a non toxic component or acomposition according to embodiment 52.

59. Use of the polypeptide according to any one of embodiments 1-44 or apolypeptide identified according to embodiment 45 or a polypeptideprepared according to embodiments 46-47 or the nucleotide sequenceaccording to embodiment 48 or the vector according to embodiment 49 orthe cell according to embodiment 50 or the organism according toembodiment 51 admixed with a non toxic component or a compositionaccording to embodiment 52 in a method of modifying plant materials.

60. Use of the polypeptide according to any one of embodiments 1-44 or apolypeptide identified according to embodiment 45 or a polypeptideprepared according to embodiments 46-47 or the nucleotide sequenceaccording to embodiment 48 or the vector according to embodiment 49 orthe cell according to embodiment 50 or the organism according toembodiment 51 admixed with a non toxic component or a compositionaccording to embodiment 52 in any one or more independently selectedfrom: baking, processing cereals, starch liquefaction, production ofBio-ethanol from cellulosic material, animal feed, in processing wood,enhancing the bleaching of wood pulp, and as a cleaning composition.

61. A polypeptide or fragment thereof substantially as hereinbeforedescribed with reference to the Examples and drawings.

62. A method substantially as hereinbefore described with reference tothe Examples and drawings.

63. A composition substantially as hereinbefore described with referenceto the Examples and drawings.

64. A use substantially as hereinbefore described with reference to theExamples and drawings.

REFERENCES

-   Collins, T., Gerday, C. and Feller, G. (2005) FEMS Microbiol Rev.,    29 (1), 3-23.-   Courtin, C., Roelants, A. and Delcour, J. (1999).    Fractionation-reconstitution experiments provide insight into the    role of endoxylanases in bread-making. Journal of Agricultural and    Food Chemistry. 47. 1870-1877.-   Coutinho, P. M. and Henrissat, B. (1999) Carbohydrate-Active Enzymes    server at URL: afmb.cnrs-mrs.fr/CAZY/.-   D'Appolonia, B. L. and MacArthur, L. A. (1976). Comparison of bran    and endosperm pentosans in immature and mature wheat. Cereal    Chem. 53. 711-718.-   Debyser, W. and Delcour, J. A. (1998). Inhibitors of cellolytic,    xylanolytic and β-glucanolytic enzymes. WO 98/49278.-   Hazlewood, G. P. and Gelbert, H. J. (1993). Recombinant xylanases.    PCT application. WO 93/25693.-   Henrissat, B. (1991) Biochem. J. 280, 309-316.-   Ingelbrecht, J. A., Verwimp, T. and Delcour, J. A. (1999).    Endoxylanases in durum wheat semolina processing: solubilisation of    arabinoxylans, action of endogenous inhibitors and effects on    rheological properties. J. Agri. Food Chem.-   Jacobsen, T. S., Heldt-Hansen, H. P., Kofod, L. V., Bagger, C. and    Müllertz, A. (1995). Processing plant material with xylanase. PCT    application. WO 95/23514.-   Kormelink, F. J. M. (1992). Characterisation and mode of action of    xylanases and some accessory enzymes. Ph.D. Thesis, Agricultural    University Wageningen, Holland (175 pp., English and Dutch    summaries).-   Lever, M. (1972). A new reaction for colorimetric determination of    carbohydrates. Analytical Biochemistry. 47, 273-279.-   McLauchlan, R., Garcia-Conesa, M. T., Williamson, G., Roza, M.,    Ravestein, P. and MacGregor, A. W. (1999a). A novel class of protein    from wheat which inhibits xylanases. Biochem. J. 338. 441-446.-   McLauchlan, R, Flatman, R et al (1999) Poster Presentation from    meeting at University of Newcastle (1999) Apr. 11^(th)-Apr. 17^(th).    Xylanase inhibitors, a novel Class of proteins from cereals.-   Montgomery, R. and Smith, F. (1955). The Carbohydrates of the    Gramineae. VIII. The constitution of a water soluble hemicellulose    of the endosperm of wheat (Triticum vulgare). J. Am. Chem. Soc. 77.    3325-3328.-   Paice, M. G., Bourbonnais, R., Desrochers, M., Jurasek, L. and    Yaguchi, M. (1986): A Xylanase Gene from Bacillus subtilis:    Nucleotide Sequence and Comparison with B. pumilus Gene. Arch.    Microbiol. 144, 201-206.)-   Rouau, X. (1993). Investigations into the effects of an enzyme    preparation from baking on wheat flour dough pentosans. J. Cereal    Science. 18.145-157.-   Rouau, X., El-Hayek, M-L. and Moreau, D. (1994). Effect of an enzyme    preparation containing pentosanases on the bread-making quality of    flour in relation to changes in pentosan properties. J. Cereal    Science. 19.259-272.-   Slade, L., Levine, H., Craig, S., Arciszewski, H. and Saunders, S.    (1993). Enzyme treated low moisture content comestible products.    U.S. Pat. No. 5,200,215 by Nabisco.-   Soerensen, J. F. and Sibbesen, O. (1999). Bacterial xylanase. UK A    9828599.2.

The invention claimed is:
 1. A polypeptide having xylanase activity andcomprising an amino acid sequence, said amino acid sequence having atleast 88% identity with SEQ ID No. 1, and which polypeptide has an aminoacid substitution in position 110, with any one different amino acidresidue selected from the group consisting of: asparagine (N), glutamicacid (E), tryptophan (W), alanine (A) and cysteine (C), and one or moreamino acid substitutions selected from the group consisting of: 11F,12F, 122D, 113A, 13Y, 54Q, 54W, 113D, 141Q, 175L, 122F, 34K, 99Y, 104W,154R, 159D, 175K, 81I, 166F, 162E, 162D, 164F, 114D, 114Y, 114F, 118V,175K, 77L, 77M, 77S, 77V, and 77Y, the position(s) being determined asthe corresponding position(s) of B. subtilis amino acid sequence shownas SEQ ID No.
 1. 2. A polypeptide according to claim 1 having xylanaseactivity and comprising an amino acid sequence, said amino acid sequencehaving at least 90% identity with SEQ ID No. 1 and which polypeptide hasan amino acid modification in position 110, wherein said position 110 isdetermined as the position corresponding to position 110 of B. subtilisxylanase sequence shown as SEQ ID No. 1 by alignment.
 3. The polypeptideaccording to claim 2, wherein said polypeptide has at least 95% identitywith SEQ ID No.
 1. 4. The polypeptide according to claim 1, having aβ-jelly roll fold.
 5. The polypeptide according to claim 1, wherein theamino acid modification in position 110 is a substitution to alanine. 6.The polypeptide according to claim 1, having a total number of aminoacids of less than
 250. 7. A composition comprising the polypeptideaccording to claim 1 admixed with a non-toxic component.